Minoxidil Adjuvant Therapies

ABSTRACT

Compositions and methods are disclosed herein for inducing (up-regulating) the expression of sulfotransferases in the hair follicles, e.g., the scalp. Increasing sulfotransferase is beneficial for metabolizing pro-drugs that require sulfonation to be activated, e.g., minoxidil sulfate is the active metabolite of minoxidil. A method for combining the compositions described herein with topical minoxidil to enhance minoxidil treatment for androgenetic alopecia is described. Additional methods and compositions include the use of retinoid X receptor agonists, retinoic acid receptor agonists, and nuclear receptor agonists in an RXR-NR heterodimer. Additional methods and compositions include the use of a topical solution containing an alkalinizing agent or an alkalinizing agent used with a penetration enhancer for up-regulating the sulfonating capacity of hair bearing skin, hair follicles, and/or keratinocyte cells. In addition, compositions and methods for increasing or decreasing the growth rate of hair follicles is disclosed by altering the intracellular pH. Additionally, methods for preparing a stable liposomal containing solution are described.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part application of U.S. Ser. No.16/747,685, filed on Jan. 21, 2020, the U.S. Ser. No. 16/747,685application being related to and claiming the benefit of MINOXIDILADJUVANT THERAPIES, filed on Oct. 4, 2019 and issued application Ser.No. 16/593,577, the Ser. No. 16/593,577 application being related to andclaiming the benefit of MINOXIDIL ADJUVANT THERAPIES, filed on Oct. 5,2018 and issued Application No. 62/741,990, MINOXIDIL ADJUVANTTHERAPIES, filed on Nov. 6, 2018 and issued Application No. 62/756,293,MINOXIDIL ADJUVANT THERAPIES, filed on Feb. 1, 2019 and issuedApplication No. 62/800,065, MINOXIDIL ADJUVANT THERAPIES, filed on May17, 2019 and issued Application No. 62/849,598, and MINOXIDIL ADJUVANTTHERAPIES, filed on Sep. 4, 2019 and issued Application No. 62/895,627,the entire contents of each being incorporated herein by reference intheir entirety.

FIELD

The present invention relates to methods and compositions that inducethe expression of sulfotransferase in the hair follicle. Topicalcompositions containing agents that bind to the transcription factorsthat mediate the xenobiotic response in cells (e.g., PDX and CAR) aredescribed. Up-regulation of sulfotransferase is beneficial to activatecertain prodrugs that require sulfonation to become activated. One suchdrug is minoxidil used to treat androgenetic alopecia. The presentinvention is directed to methods for treating, reducing or preventingalopecia and other hair loss disorders by applying a sulfotransferaseinducing composition on the scalp prior to treatment with minoxidil.Additional embodiments relate to compositions and kits that diagnose andcontrol hair follicle stem cell differentiation. Methods andcompositions are described that modify hair follicle stem cellsintracellular pH thus controlling hair follicle stem celldifferentiation. In some instances, increase of hair follicle stem cellsintracellular pH induces and/or increases the sulfotrasnfrease enzymaticactivity in hair follicle cells. Induction of the sulfotransferaseenzyme in hair follicles increases the sulfonation capacity ofminoxidil; thus, increasing the response level to oral and topicalminoxidil in the treatment of alopecia.

BACKGROUND

In 1988 the US FDA approved 2% topical minoxidil solution as an OTC drugfor the treatment of androgenetic alopecia (AGA). Since the FDAapproval, minoxidil has become the mainstay therapy for AGA. However,the effectiveness of minoxidil in the general population is low, only39% of patients respond to the drug (See Olsen E A, Whiting D, BergfeldW, Miller J, Hordinsky M, Wanser R, et al. A multicenter, randomized,placebo-controlled, double-blind clinical trial of a novel formulationof 5% minoxidil topical foam versus placebo in the treatment ofandrogenetic alopecia in men. J Am Acad Dermatol. 2007 57(5): 767-74).In the pivotal study submitted to the US FDA in support of the efficacyof the 5% topical minoxidil foam, no subjects had great improvement, 8%of the subjects had a moderate improvement, and 31% of the subjects hada slight improvement (See US FDA Application 21-812 Medical Review).

Minoxidil is a pro-drug converted to its active form, minoxidil sulfate,by sulfotransferase enzymes present in the outer root sheath (ORS) ofhair follicles (See Buhl A E, Waldon D J, Baker C A, Johnson G A.Minoxidil sulfate is the active metabolite that stimulates hairfollicles. J Invest Dermatol. 1990 November; 95(5):553-7). It has beendemonstrated that the activity of sulfotransferase in the ORS determinesthe clinical response to minoxidil (See Goren A, Castano J A, McCoy J,Bermudez F, Lotti T. Novel enzymatic assay predicts minoxidil responsein the treatment of androgenetic alopecia. Dermatol Ther. 2014;27(3):171-3). Sulfotransferase enzymes are expressed in abundance in thehuman liver. In the human liver, sulfotransferase is part of the PhaseII enzymatic system that reduces xenobiotic toxicity (See Jancova P,Anzenbacher P, Anzenbacherova E. Phase II drug metabolizing enzymes.Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub. 2010;154(2):103-16).

Xenobiotics are extrinsic chemical substances (e.g., drugs), which maybe present in human body (See Croom E. Metabolism of xenobiotics ofhuman environments. Prog Mol Biol Transl Sci 2012; 112: 31-88). They areincluded into specific metabolic pathways evolved to mitigate toxicityto an organism. Xenobiotic metabolism includes several pathways designedto modify chemical structure and decrease the toxicity of the compounds.Although, in some instances, the intermediates in xenobiotic metabolismmay themselves cause toxic effects (See Bunchorntavakul C, Reddy K R.Acetaminophen (APAP or N-Acetyl-p-Aminophenol) and Acute Liver Failure.Clinics in Liver Disease 2018; 22(2): 325-346). Xenobiotic metabolism isdivided into 3 phases, organized to modify lipophilic compounds intohydrophilic conjugates that can more readily be excreted. In Phase I,lipophilic xenobiotic molecules are metabolized by enzymes such ascytochrome P450 oxidases, which introduce polar groups and provide sitesfor downstream conjugation reactions. Phase I reactions are mainlylocalized in the liver. In Phase II, conjugating enzymes interact withmetabolites produced by Phase I enzymes and eliminate them through bothpassive and active transport. Conjugating enzymes include a large groupof broad-specificity transferases, with glutathione S-transferases asthe most important representatives (See Jakoby W B, Ziegler D M. Theenzymes of detoxication. J Biol Chem 1990; 265(34): 20715-20718). Afterconjugation, any xenobiotic conjugates or their metabolites noteliminated in Phase II are further processed and eliminated in Phase IIIby transporter proteins.

Several Phase I and II metabolizing enzymes are known to be inducible byboth endogenous and xenobiotic molecules. The most widely studieddrug-metabolizing enzyme, by far, is the cytochrome P450 family. Many ofthe members of the cytochrome P450 family are inducible via nuclearreceptor mediated induction. For example, cytochrome family 1 genes areup-regulated by the aryl hydrocarbon receptor (AhR) after it bindsaromatic hydrocarbon ligands (See Gonzalez F J, Liu S Y, Yano M.Regulation of cytochrome P450 genes: molecular mechanisms.Pharmacogenetics 1993; 3(1): 51-57). Similarly, sulfotransferases havebeen demonstrated to be regulated by endogenous hormones (SeeRunge-Morris M, Kocarek T A, Falany C N. Regulation of the cytosolicsulfotransferases by nuclear receptors. Drug metabolism reviews 2013;45(1): 15-33) and xenobiotics (See Runge-Morris M, Kocarek T A.Regulation of sulfotransferases by xenobiotic receptors. Curr Drug Metab2005; 6(4): 299-307).

Minoxidil sulfate is required for both the promotion of hair regrowthand the vasodilatory effects of minoxidil. Sulfotransferase enzymes arelocated in both the skin and the liver and are important Phase IIxenobiotic metabolizing enzymes for a number of phenolic moleculesincluding minoxidil (See Nimmagadda D, Cherala G, Ghatta S. Cytosolicsulfotransferases. Indian J Exp Biol 2006; 44(3): 171-182).

Xenobiotic-metabolizing enzymes are important for the metabolism,elimination or detoxification of xenobiotics. Various nuclear receptorsincluding aryl hydrocarbon receptor (AhR) and constitutive androstanereceptor (CAR) regulate the gene expressions of Xenobiotic-metabolizingenzymes (See Xu, C., Li, C. Y., Kong, A. N., 2005. Induction of phase I,II and III drug metabolism/transport by xenobiotics. Arch. Pharm. Res.28, 249-268). Upon ligand binding, AhR forms a heterodimer with the AhRnuclear translocator (Arnt), and the AhR-Arnt complex binds to specificxenobiotic responsive elements and activates a battery of genesincluding members of cytochrome P450 family 1 (CYP1), such as CYP1A1,CYP1A2, CYP1B1, and UDP-glucuronosyltransferases (UGT) 1A1, 1A6, 1A7 and1A9 involved in the detoxification and elimination of xenobiotics aswell as certain endogenous steroids. CAR and pregnane X receptor (PXR)are nuclear receptors that form functional heterodimers with theretinoid X receptor (RXR) (See Honkakoski, P., Sueyoshi, T., Negishi,M., 2003. Drug-activated nuclear receptors CAR and PXR. Ann. Med. 35,172-182). CAR and PXR are responsible for the xenobiotic-mediatedinduction of many genes including CYP1A, 2B, 2C and 3A families, UGT1A1and 1A3, and sulfotransferase (SULT) 1A1 and 2A1 (See Handschin, C.,Meyer, U. A., 2003. Induction of drug metabolism: the role of nuclearreceptors. Pharmacol. Rev. 55, 649-673 and Maglich, J. M., Stoltz, C.M., Goodwin, B., Hawkins-Brown, D., Moore, J. T., Kliewer, S. A., 2002.Nuclear pregnane x receptor and constitutive androstane receptorregulate overlapping but distinct sets of genes involved in xenobioticdetoxification. Mol. Pharmacol. 62, 638-646). Additionally,3′-phosphoadenosine 5′-phosphosulfate (PAPS) synthase (PAPSS), whichcatalyzes the biosynthesis of PAPS, which serves as the universalsulfonate donor compound for all sulfotransferase reactions is regulatedby PXR and CAR (See Owen B M, Milona A, van Mil S, Clements P, Holder J,Boudjelal M, Cairns W, Parker M, White R, Williamson C. Intestinaldetoxification limits the activation of hepatic pregnane X receptor bylithocholic acid. Drug Metab Dispos. 2010 January; 38(1):143-9. andAlnouti Y, Klaassen C D. Regulation of sulfotransferase enzymes byprototypical microsomal enzyme inducers in mice. J Pharmacol Exp Ther.2008 February; 324(2):612-21).

Many compounds have been reported to bind to nuclear factors and induceor suppress the expression of xenobiotic-metabolizing enzymes. For acomprehensive list See A. Parkinson, B. W. Ogilvie, D. B. Buckley, F.Kazmi, M. Czerwinski, O. Parkinson, Biotransformation of xenobiotics,in: C. Klaassen (Ed.), Casarett & Doull's Toxicology, The Basic Scienceof Poisons, McGraw-Hill Education, New York, N.Y., USA, 2013, pp.185-366.

Additionally, altering intracellular or extracellular pH is an importantregulatory mechanism, which can influence cellular function and lead tocell differentiation in a range of stem cells (See Charruyer A,Ghadially R. Influence of pH on Skin Stem Cells and TheirDifferentiation. Curr Probl Dermatol. 2018; 54: 71-78). Celldifferentiation is associated with the altered expression of manyproteins including xenobiotic-metabolizing enzymes. Specifically,increased sulfotransferase is a marker for keratinocyte differentiation(See, Johnson G A, Baker C A, Knight K A. Minoxidil sulfotransferase, amarker of human keratinocyte differentiation. J Invest Dermatol. 1992May; 98(5): 730-3).

All studies to date exploring nuclear factors ability to induce orsuppress the expression of xenobiotic-metabolizing enzymes have beenconducted in cultured liver cells, cultured colon cells, or in mice. Inorder to determine the induction of xenobiotic-metabolizing enzymes inscalp tissues new methodologies will need to be employed. One suchtechnology is a colorimetric assay for detecting sulfotranseferase inplucked hair samples described in U.S. Pat. No. 8,691,518, which isincorporated herein in its entirety by reference.

Many methodologies exist for increasing the dermal penetration of activeingredients through the stratum corneum (SC). For example, the use ofpenetration enhancing chemical agents have been described (See TrommerH, Neubert R H. Overcoming the stratum corneum: the modulation of skinpenetration. A review. Skin Pharmacol Physiol. 2006; 19(2):106-21).However, many of these agents alter the structure of the SC aggressivelyand can lead to irreversible damage to the SC. An alternative approachto the use of SC modifying agents is the use of encapsulation techniques(See Tiwari N, Osorio-Blanco E R, Sonzogni A, Esporrín-Ubieto D, Wang H,Calderón M. Nanocarriers for Skin Applications: Where Do We Stand? AngewChem Int Ed Engl. 2022 Jan. 17;61(3):e202107960). Such techniquessurround the active ingredients with carrier molecules sharing similarmorphology to cellular membranes that interact more gently with the SCand can be used to deliver large payloads. Such systems are morebiocompatible and offer advantages such as biodegradability and lowtoxicity. Many examples of such encapsulation techniques have beendescribed—for example, liposomes, micelles, nanogels, lipidnanoparticles, selenium nanoparticles, bilosomes, dendrimers and carbonnanotubes. Unfortunately, the use of encapsulation technologiesintroduces additional challenges to the production of a stable topicalformulas. Many of the encapsulating systems used are susceptible tohydrolysis, oxidation, and aggregation or breakdown of the macroencapsulation structure. As such, novel formulation technologies arerequired to successfully implement a stable and operational topicalformula containing encapsulated actives.

SUMMARY

Compositions and methods are disclosed herein for inducing(up-regulating) the expression of sulfotransferases in hair bearingskin, hair follicles, and/or keratinocyte cells, e.g., the scalp. Inaddition, compositions, methods, and kits are disclosed herein forcontrolling hair follicle stem cell differentiation. In some instances,the compositions and methods induce (up-regulate) the expression oractivity of sulfotransferases in hair bearing skin, hair follicles,and/or keratinocyte cells. Increasing sulfotransferase is beneficial formetabolizing pro-drugs that require sulfonation to be activated, e.g.,minoxidil sulfate is the active metabolite of minoxidil. Combining thecomposition described herein with topical minoxidil would be a moreefficacious treatment for androgenetic alopecia. For example,embodiments of the methods and compositions disclosed herein can be usedto increase the metabolism of minoxidil (which can result in theincrease of bioavailable minioxidil sulfate) in hair folicles ofpatients suffering from a form of alopecia. Additionally, an example ofa topical composition, a shampoo, used to increase sulfotransferase inthe scalp is described. Furthermore, methodologies for creating a stabletopical formula containing an encapsulated active ingredient aredescribed. In other instances, the compositions and methods alter thehair follicle stem cells pH and thus slows hair growth. This can bebeneficial to slow normal hair growth so people could shave lessfrequently.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is tabulated assessment of data and a global photographyassessment graph related to a study conducted to evaluate an embodimentof AB-103 as an adjuvant therapy.

FIG. 2 is tabulated sulfotransferase enzyme activity data and a meanchange in follicular sulfotransferase activity graph for a studyconducted to evaluate an embodiment of AB-103 for up-regulatingminoxidil sulfotransferase enzymes.

FIG. 3 is tabulated assessment of data and an expert assessment graphrelated to another study conducted to evaluate an embodiment of AB-103as an adjuvant therapy.

FIG. 4 is tabulated assessment of data and an assessment graph relatedthe mean change in target area hair growth for another study conductedto evaluate an embodiment of AB-103 as an adjuvant therapy.

FIG. 5 is a table showing results for PHi of HFSC niche in hairfollicles.

FIGS. 6-8 are tables showing results of a study assessing the ability tochange HFSC pHi.

FIG. 9 shows results of % increase in activity per hour at pH 9.5 fortwo subjects.

DETAILED DESCRIPTION

Androgenetic alopecia (AGA) is a common dermatological conditionaffecting approximately 50% of the population by the age of 50.Currently, the only drug approved by the US Food and Drug Administration(FDA) for the treatment of AGA in both men and women is topicalminoxidil. Clinical trials have demonstrated that following 16 weeks of5% minoxidil therapy approximately 30-40% of patients regrow hair.

While the exact mechanism of action of minoxidil in the treatment of AGAis not completely understood, research has demonstrated that minoxidilsulfate is the active compound that stimulates hair follicles. Minoxidilis converted to its active form, minoxidil sulfate, in the outer rootsheath of the hair follicle by endogenous sulfotransferase enzymesutilizing 3′-phosphoadenosine 5′-phosphosulfate (PAPS). PAPS is producedin cells utilizing 3′-phosphoadenosine 5′-phosphosulfate synthase(PAPSS). Several studies have reported a correlation betweensulfotransferase activity in plucked hair follicles and minoxidilresponse for AGA patients. In theory, increasing sulfotransferase orPAPS in the scalp would increase the likelihood that a subject willrespond to topical minoxidil (thereby increasing the efficacy of atopical minoxidil); however, this has not been demonstrated in clinicalstudies. Further, in medicine, the induction of a deficient enzymefrequently does not result in a clinical benefit. For example, thepro-drug acyclovir used for the treatment of HSV is activated by thehuman thymidine kinase enzyme; however, while the induction of thethymidine kinase enzyme in-vitro activates acyclovir, in human studiesthymidine kinase enzyme induction does not convert non-responders toacyclovir into responders. As noted herein, minoxidil compositions canbe used to treat forms of alopecia. Thus, increasing the efficacy ofminoxidil (which can involve converting androgenetic alopecia patientswho are non-responders into responders to minoxidil) can improve hairgrowth (inclusing hair diameter) of subjects using topical minoxidil. Inaddition, methods and compositions disclosed herein for increasing theefficacy of minoxidil for treatment of forms of alopecia can acceleratehair growth of subjects using topical minoxidil.

Biological sulfation is the conversion of the very stable oxy-anionsulfate to the high-energy sulfate donor3′-phospho-adenosine-5′-phosphosulphate (PAPS). Sulfation of a varietyof biomolecules depend on availability of the precursor PAPS, which israte-limiting. In mammals, PAPS is synthesized in two steps by abi-functional enzyme called PAPS synthetase (PAPSS). The synthesis ofPAPS from inorganic sulfate and ATP is catalyzed by PAPSS. The sourcesof inorganic sulfur in nature are broad but include cysteine,1-cysteine, hydrogen sulfide, elemental sulfur, sulfite, thiosulfate,and various polythionates (e.g., tetrathionate).

In one embodiment, the invention concerns up-regulating the sulfonatingcapacity of hair bearing skin by applying a topical solution containinga source of inorganic sulfur to increase the concentration of PAPS.Examples of sources of inorganic sulfur include but are not limited tocysteine, 1-cysteine, hydrogen sulfide, elemental sulfur, sulfite,thiosulfate, and various polythionates (e.g., tetrathionate).Additionally, a sulfate salt may be used, for example magnesium sulfateor sodium sulfate.

As used herein, the terms “prevent” or “prevention” and otherderivatives of the words, when used in reference to alopecia, e.g.,androgenetic alopecia, refer to a reduced likelihood of alopecia in anindividual receiving a given treatment relative to that of a similarindividual at risk for alopecia but not receiving that treatment. Assuch, the terms “prevent” and “prevention” encompass a treatment thatresults in a lesser degree of alopecia, e.g., androgenetic alopecia,than would be otherwise expected for a given individual. Efficacy forprevention of alopecia, e.g., androgenetic alopecia, can be establishedthrough controlled studies, e.g., in which a subject is administered atreatment (e.g., a topical treatment) and another subject isadministered a placebo. Under these circumstances, if the subjecttreated with the topical treatment undergoes less hair loss over timerelative to the subject receiving the placebo, e.g., at least 5% less,at least 10% less, at least 15% less, at least 20% less, at least 25%less, at least 30% less, at least 35% less, at least 40% less, at least45% less, at least 50% less or beyond, the treatment is effective forthe prevention of alopecia, e.g., androgenetic alopecia.

As used herein, the terms “treat,” “treatment,” or “treating” refer totherapeutic treatments, wherein the object is to reverse, alleviate,ameliorate, inhibit, slow down or stop the progression or severity of adisease or condition, e.g., androgenetic alopecia or other form ofalopecia. The term “treating” includes reducing or alleviating at leastone adverse effect or symptom of a disease or condition, e.g.,androgenetic alopecia or other form of alopecia. Treatment is generally“effective” if one or more symptoms are reduced. Alternatively,treatment is “effective” if the progression of a disease is reduced orhalted. That is, “treatment” includes not just the improvement ofsymptoms, but also a cessation of, or at least slowing of, progress orworsening of symptoms compared to what would be expected in the absenceof treatment. Beneficial or desired clinical results include, but arenot limited to, alleviation of one or more symptom(s), diminishment ofextent of disease, stabilized (i.e., not worsening) state of disease,delay or slowing of disease progression, amelioration or palliation ofthe disease state, remission (whether partial or total), and/ordecreased mortality. For example, treatment is considered effective ifthe extent or amount of hair loss is reduced, or the progression of hairloss is slowed or halted. The term “treatment” of a disease alsoincludes providing relief from the symptoms or side-effects of thedisease (including palliative treatment).

As used herein the term “comprising” or “comprises” is used in referenceto compositions, methods, etc. refers to component(s) or method stepsthat are present in the method or composition, yet allows for thecomposition, method, etc. to also include unspecified elements.

The term “consisting of” refers to compositions, methods, and respectivecomponents thereof as described herein, which are exclusive of anyelement not recited in that description of the embodiment.

As used herein the term “consisting essentially of” refers to thoseelements required for a given embodiment. The term permits the presenceof elements that do not materially affect the basic and novel orfunctional characteristic(s) of that embodiment.

As used herein the term “alopecia” refers to all forms of hair loss inmen and women including but not limited to traction alopecia,androgenetic alopecia, male pattern baldness (MPB), female pattern hairloss (MIL), alopecia areata, alopecia universalis, telogen effluvium,chemotherapy induced alopecia, hair shedding, eybrow hair loss, beardhair loss, hair thinning, etc. The term permits the presence of elementsthat do not materially affect the basic and novel or functionalcharacteristic(s) of that embodiment.

As used herein the term “alkalinizing agent” refers to all agents thateither: (i) directly increase the intracellular pH (ii) directlyincrease the intracellular pH by activating or inhibiting the variousion carriers that regulate cellular pH (iii) upregulate or downregulatethe various ion carriers; or (iv) increase the intracellular pH bychanging the extracellular pH. The term permits the presence of elementsthat do not materially affect the basic and novel or functionalcharacteristic(s) of that embodiment.

As used herein the term “acidifying agent” refers to all agents thateither: (i) directly decrease the intracellular pH (ii) directlydecrease the intracellular pH by activating or inhibiting the variousion carriers that regulate cellular pH (iii) upregulate or downregulatethe various ion carriers; or (iv) decrease the intracellular pH bychanging the extracellular pH. The term permits the presence of elementsthat do not materially affect the basic and novel or functionalcharacteristic(s) of that embodiment.

As used herein the term “ion carriers” refers to all cellular ioncarriers that either increase or decrease the intracellular pH. In thecontext of this application ion carriers can be referred to as protonpumps. The term permits the presence of elements that do not materiallyaffect the basic and novel or functional characteristic(s) of thatembodiment.

The singular terms “a,” “an,” and “the” include plural referents unlesscontext clearly indicates otherwise. Similarly, the word “or” isintended to include “and” unless the context clearly indicatesotherwise. Although methods and materials similar or equivalent to thosedescribed herein can be used in the practice or testing of thisdisclosure, suitable methods and materials are described below. Theabbreviation, “e.g.” is derived from the Latin exempli gratia, and isused herein to indicate a non-limiting example. Thus, the abbreviation“e.g.” is synonymous with the term “for example.”

Various aspects of the technology described measuring the sulfonatingability of a hair bearing skin, hair follicle, and/or keratinocyte cell.An increase in the sulfonating ability of a hair bearing skin, hairfollicle, and/or keratinocyte cell can be interpreted to mean thatenzymes or substrates required for this reaction have been increased inconcentration, i.e., an increase in substrates will lead to an increasein reaction products (Le Chatelier's principal). For example, increasingthe available sulfotranseferase will increase the sulfonating ability ofa hair bearing skin, hair follicle, and/or keratinocyte cell. Similarly,increasing the available PAPS or PAPSS (which produces PAPS) willincrease the sulfonating ability of a hair bearing skin, hair follicle,and/or keratinocyte cell.

Measurement of the sulfonating ability of a hair bearing skin, hairfollicle, and/or keratinocyte cell or a hair follicle can be performed,if necessary, via a colorimetric assay adapted for that purpose.Examples are described in, e.g., Goren A, Shapiro J, Roberts J, McCoy J,Desai N, Zarrab Z, Pietrzak A, Lotti T. Clinical utility and validity ofminoxidil response testing in androgenetic alopecia. Dermatol Ther 2015:28(1): 13-16, which is incorporated herein in its entirety by reference.Briefly, plucked anagen hairs are collected from the scalp and inspectedvisually for an intact bulb. Suitable hairs are trimmed to a length of˜1 cm and immersed, bulb first, in 100 μL of an assay solutioncontaining 50 mM phosphate buffer (pH8), 5 mM potassium p-nitrophenylsulfate, 20 μM adenosine 3′,5′-diphosphate, 100 μM minoxidil and 5 mMMgCl2. Hairs are allowed to react with the solution for 24 hours at roomtemperature. After incubation, hairs are removed and the opticalabsorbance of the solution at 405 nm is determined with aspectrophotometer (e.g., Shimadzu UV-1700, Kyoto, Japan) using a singlescan and 1 cm path length.

Increased intracellular pH is necessary for adult epithelial andembryonic stem cell differentiation. Various aspects of the inventiondescribe the increased or decreased rate of hair follicle stem cells(HFSC), rate of differentiation, and/or proliferation. An increase inintracellular pH (pHi) can be used to increase the rate of hair folliclestem cells (HFSC) rate of differentiation and/or proliferation.Similarly, a decrease in intracellular pH (pHi) can be used to decreasethe rate of hair follicle stem cells (HFSC) rate of differentiationand/or proliferation. In one aspect of the invention, pHi can be changedby changing the extracellular pH (pHe).

Applicants disclose herein methods to treat or prevent various forms ofalopecia, e.g. female pattern hair loss or androgenetic alopecia. Themethod includes the use of a topical composition applied to the scalpthat up-regulates the sulfonating capacity of the hair follicle. Themethod further includes the use of topical minoxidil applied subsequentto a topical composition applied to the scalp that up-regulates thesulfonating capacity of the hair follicle.

Additionally, applicant discloses herein methods for slowing hairgrowth. The methods include application of an acidfying agent to theHFSC niche subsequently reducing the HFSC pHi.

In one embodiment, the invention concerns up-regulating the sulfonatingcapacity of hair bearing skin, hair follicles, and/or keratinocyte cellsby applying a topical solution containing an alkalinizing agent thatwill raise the intracellular pH of cells in the outer root sheath of thehair follicle. Examples of alkalinizing agents include, but are notlimited to, sodium bicarbonate, sodium citrate, potassium citrate,calcium carbonate, sodium lactate, and calcium acetate, carbicarb,sodium citrate/citric acid.

In one embodiment, the invention concerns up-regulating the sulfonatingcapacity of hair bearing skin, hair follicles, and/or keratinocyte cellsby applying a topical solution containing an alkalinizing agent thatwill raise the extracellular pH of cells in the outer root sheath of thehair follicle.

In one embodiment, the invention concerns up-regulating the sulfonatingcapacity of hair bearing skin, hair follicles, and/or keratinocyte cellsby applying a topical solution containing an alkalinizing agent with apenetration enhancer. Examples of penetration enhancers include, but arenot limited to, alcohols, glycols (e.g., diethylene glycol andtetraethylene glycol), fatty acids (e.g., lauric acid, myristic acid andcapric acid), fatty esters, fatty ethers, cyclodextrines, occlusiveagents, surface active agents, dimethylaminopropionic acid derivatives,terpenes, sulfoxides, cyclic ethers, amides, and amines. Other examplesof penetration enhancers can include sulphoxides (such asdimethylsulphoxide, DMSO, decylmethalsulfoxide), Azones (e.g.,1-dodecylazacycloheptan-2-one, laurocapran, or laurocapram),pyrrolidones (e.g., 2-pyrrolidone, 2P, N-methylpyrrilidone,N-methyl-2-pyrrolidone, NMP, 1-propyl-3-dodecyl-2-pyrrolidone,1-butyl-3-dodecyl-2-pyrrolidone), alcohols and alkanols (ethanol, ordecanol), glycols (e.g., propylene glycol), surfactants (e.g.,polyoxyethylene-2-oleyl ether, polyoxy ethylene-2-stearly ether, sodiumdodecyl sulfate, SDS, sodium lauryl sulfate, SLS), Oxazolidinones (e.g.,4-decyloxazolidin-2-one), urea, 2-(1-nonyl)-1,3-dioxolane, and terpenes.Additional examples of penetration enhancers can include polyesternanosponges, liposomes, phospholipids, cyclopentadecalactone,pentadecalactone, SNAC, salcaprozate sodium SodiumN-[8-(2-hydroxybenzoyl) amino]caprylate, CNAC, 5-CNAC,8-(N-2-hydroxy-5-chloro-benzyl)-amino-caprylic acid, sodium caprate,glyceryl triglyceride, and peptides.

In one embodiment, the invention concerns up-regulating the sulfonatingcapacity of hair bearing skin, hair follicles, and/or keratinocyte cellsby applying a topical solution containing an alkalinizing agent with adevice designed to increase transdermal penetration. Examples of devicesdesigned to increase transdermal penetration include micro-needle arraysand iontophoretic patches.

In one embodiment, the invention concerns up-regulating the sulfonatingcapacity of hair bearing skin, hair follicles, and/or keratinocyte cellsby raising the intracellular pH of stem cells in the outer root sheathof a hair follicle. In one embodiment, intracellular pH of stem cellsmay be alkalinized by application of a topical proton pump agonist. Inanother embodiment, intracellular pH of stem cells may be alkalinized byapplication of a topical proton pump agonist.

In one embodiment, the invention concerns treatment of alopecia byinducing HFSC differentiation and hair anagen cycle elongation byapplying a topical solution containing an alkalinizing agent that willraise the extracellular pH of cells in the outer root sheath of the hairfollicle.

In one embodiment, the invention concerns treatment of alopecia byinducing HFSC differentiation and hair anagen cycle elongation byapplying a topical solution containing an alkalinizing agent with apenetration enhancer. Examples of penetration enhancers include, but arenot limited to, alcohols, glycols (e.g., diethylene glycol andtetraethylene glycol), fatty acids (e.g., lauric acid, myristic acid andcapric acid), fatty esters, fatty ethers, cyclodextrines, occlusiveagents, surface active agents, dimethylaminopropionic acid derivatives,terpenes, sulfoxides, cyclic ethers, amides, and amines. Other examplesof penetration enhancers can include sulphoxides (such asdimethylsulphoxide, DMSO, decylmethalsulfoxide), Azones (e.g.,1-dodecylazacycloheptan-2-one, laurocapran, or laurocapram),pyrrolidones (e.g., 2-pyrrolidone, 2P, N-methylpyrrilidone,N-methyl-2-pyrrolidone, NMP, 1-propyl-3-dodecyl-2-pyrrolidone,1-butyl-3-dodecyl-2-pyrrolidone), alcohols and alkanols (ethanol, ordecanol), glycols (e.g., propylene glycol), surfactants (e.g.,polyoxyethylene-2-oleyl ether, polyoxy ethylene-2-stearly ether, sodiumdodecyl sulfate, SDS, sodium lauryl sulfate, SLS), Oxazolidinones (e.g.,4-decyloxazolidin-2-one), urea, 2-(1-nonyl)-1,3-dioxolane, and terpenes.Additional examples of penetration enhancers can include polyesternanosponges, liposomes, phospholipids, cyclopentadecalactone,pentadecalactone, SNAC, salcaprozate sodium SodiumN-[8-(2-hydroxybenzoyl) amino]caprylate, CNAC, 5-CNAC,8-(N-2-hydroxy-5-chloro-benzyl)-amino-caprylic acid, sodium caprate,glyceryl triglyceride, and peptides.

In one embodiment, the invention concerns treatment of alopecia byinducing HFSC differentiation and hair anagen cycle elongation byapplying a topical solution containing an alkalinizing agent with adevice designed to increase transdermal penetration. Examples of devicesdesigned to increase transdermal penetration include micro-needle arraysand iontophoretic patches.

In one embodiment, the invention concerns increasing hair graft survivaland reducing shock hair loss post hair surgery by applying a topicalsolution containing an alkalinizing agent that will raise theextracellular pH of cells in the outer root sheath of the hair follicle.The application of the topical solution can be made by a sprayer ormist.

In one embodiment, the invention concerns increasing hair graft survivaland reducing shock hair loss post hair surgery by applying a topicalsolution containing an alkalinizing agent with a penetration enhancer.Examples of penetration enhancers include, but are not limited to,alcohols, glycols (e.g., diethylene glycol and tetraethylene glycol),fatty acids (e.g., lauric acid, myristic acid and capric acid), fattyesters, fatty ethers, cyclodextrines, occlusive agents, surface activeagents, dimethylaminopropionic acid derivatives, terpenes, sulfoxides,cyclic ethers, amides, and amines. Other examples of penetrationenhancers can include sulphoxides (such as dimethylsulphoxide, DMSO,decylmethalsulfoxide), Azones (e.g., 1-dodecylazacycloheptan-2-one,laurocapran, or laurocapram), pyrrolidones (e.g., 2-pyrrolidone, 2P,N-methylpyrrilidone, N-methyl-2-pyrrolidone, NMP,1-propyl-3-dodecyl-2-pyrrolidone, 1-butyl-3-dodecyl-2-pyrrolidone),alcohols and alkanols (ethanol, or decanol), glycols (e.g., propyleneglycol), surfactants (e.g., polyoxyethylene-2-oleyl ether, polyoxyethylene-2-stearly ether, sodium dodecyl sulfate, SDS, sodium laurylsulfate, SLS), Oxazolidinones (e.g., 4-decyloxazolidin-2-one), urea,2-(1-nonyl)-1,3-dioxolane, and terpenes. Additional examples ofpenetration enhancers can include polyester nanosponges, liposomes,phospholipids, cyclopentadecalactone, pentadecalactone, SNAC,salcaprozate sodium Sodium N-[8-(2-hydroxybenzoyl) amino]caprylate,CNAC, 5-CNAC, 8-(N-2-hydroxy-5-chloro-benzyl)-amino-caprylic acid,sodium caprate, glyceryl triglyceride, and peptides.

In one embodiment, the invention concerns increasing hair graft survivaland reducing shock hair loss post hair surgery by applying a topicalsolution containing an alkalinizing agent with a device designed toincrease transdermal penetration. Examples of devices designed toincrease transdermal penetration include micro-needle arrays andiontophoretic patches.

In one embodiment, the invention concerns reducing hair or the rate ofgrowth of hair on hair bearing skin, hair follicles, and/or keratinocytecells by applying a topical solution containing an acidifying agent thatwill lower the intracellular pH of cells in the outer root sheath of thehair follicle. Examples of acidifying agents include, but are notlimited to, citric acid, ascorbic acid, vitamin C, lactic acid, aceticacid, etc.

In one embodiment, the invention concerns reducing hair or the rate ofgrowth of hair on hair bearing skin, hair follicles, and/or keratinocytecells by applying a topical solution containing an acidifying agent anda penetration enhancer.

In one embodiment, the invention concerns up-regulating the sulfonatingcapacity of hair bearing skin, hair follicles, and/or keratinocyte cellsby applying a topical solution containing an agonist of the AhR nuclearreceptor. Examples of AhR agonists include, but are not limited to,PAHs, TCDD (other PHAHs), β-naphthoflavone, indigoids, tryptophanmetabolites, omeprazole, and lansoprazole.

In one embodiment, the invention concerns up-regulating the sulfonatingcapacity of hair bearing skin, hair follicles, and/or keratinocyte cellsby applying a topical solution containing an agonist of the CAR nuclearreceptor. Examples of CAR agonists include, but are not limited to,phenobarbital, phenytoin, carbamazepine, CITCO (human), TCPOBOP (mouse),clotrimazole, Yin Zhi Wuang (many PXR agonists are also CAR agonists,and vice versa), and meclizine.

In one embodiment, the invention concerns up-regulating the sulfonatingcapacity of hair bearing skin, hair follicles, and/or keratinocyte cellsby applying a topical solution containing an agonist of the PXR nuclearreceptor. Examples of PXR agonists include, but are not limited to,amprenavir, avasimibe, bosentan, bile acids, carbamazepine, clindamycin,clotrimazole, cortisol, cyproterone acetate, dicloxacillin, efavirenz,etoposide, dexamethasone, genistein, griseofulvin, guggulsterone,guttiferone G, garcinol, Isogarcinol hyperforin (Saint John's Wort),indinavir, lovastatin, mifepristone, nafcillin, nelfinavir, nifedipine,omeprazole, paclitaxel, PCBs, phenobarbital, phthalate monoesters,5β-pregnane-3,20-dione, rifabutin, rifampin, ritonavir, saquinavir,simvastatin, spironolactone, sulfinpyrazole, TAO, tetracycline,topotecan, transnanoclor, troglitazone, verapamil, vitamin E, vitaminK2, artemisinin, PCN, LCA, cafestol, SR-12813, rifaximin, mevastatin,TO901317, Solomonsterol A, and meclizine.

In one embodiment, the invention concerns up-regulating the sulfonatingcapacity of hair bearing skin, hair follicles, and/or keratinocyte cellsby applying a topical solution containing an agonist of the PPARαnuclear receptor. In one embodiment, the invention concernsup-regulating the sulfonating capacity of hair bearing skin, hairfollicles, and/or keratinocyte cells by applying a topical solutioncontaining an alkalinizing agent or an alkalinizing agent with apenetration enhancer with an agonist of the PPARα nuclear receptor.Examples of PPARα agonists include, but are not limited to, fibrates,WY-14,643, and perfluorodecanoic acid.

In one embodiment, the invention concerns up-regulating the sulfonatingcapacity of hair bearing skin, hair follicles, and/or keratinocyte cellsby applying a topical solution containing an agonist of the Nrf2 nuclearreceptor. In one embodiment, the invention concerns up-regulating thesulfonating capacity of hair bearing skin, hair follicles, and/orkeratinocyte cells by applying a topical solution containing analkalinizing agent or an alkalinizing agent with a penetration enhancerwith an agonist of the Nrf2 nuclear receptor. Examples of Nrf2 agonistsinclude, but are not limited to, β-Naphthoflavone, oltipraz, phenolicantioxidants (e.g., BHA and BHT) and various glutathione depletors.

In one embodiment, the invention concerns up-regulating the sulfonatingcapacity of hair bearing skin, hair follicles, and/or keratinocyte cellsby applying a composition comprising an activator of the Nrf2 (Nuclearfactor erythroid 2-related factor 2) nuclear factor to a hair folliclecell and/or a keratinocyte cell. Examples of Nrf2 activators include,but are not limited to, Sulforaphane, Resveratrol, Curcumin, Quercetin,Epigallocatechin-3-gallate, Diallyl sulfide, Naringenin, Pterostilbene,Caffeic Acid Phenethyl Ester, Fisetin, Lithospermate B, Ferulic acid,Zerumbone, Carnosol, Cafestol, Ellagic acid, Eugenol, Kaempferol,β-Naphthoflavone, oltipraz, Bardoxolone methyl, RTA 408, CDDO-Im,Bardoxolone, Danshensu, CDDO-EA, Mangiferin, Acetylcysteine, also knownas N-acetylcysteine (NAC), butylated hydroxytoululene, BHA (butylatedhydroxyanisole), BHT (butylated hydroxytoluene), phenolic antioxidants(e.g., BHA and BHT) and various glutathione depletors.

In one embodiment, the invention concerns up-regulating the sulfonatingcapacity of hair bearing skin, hair follicles, and/or keratinocyte cellsby applying a composition comprising an inducer of the Nrf2 nuclearfactor (encoded by the NFE2L2 gene) to a hair follicle cell and/or akeratinocyte cell, i.e., agents that increase the transcription ofNFE2L2. Examples of inducers are 4-Hydroxyphenylacetic acid andacetylation via p300/CBP.

In one embodiment, the invention concerns up-regulating the sulfonatingcapacity of hair bearing skin, hair follicles, and/or keratinocyte cellsby applying a composition comprising an agent that disassociates Nrf2from Keap1 (Kelch-like ECH-associated protein 1) to a hair follicle celland/or a keratinocyte cell. The agent can be selected to disassociateNrf2 from Keap1 by alkylation of Keap1, or some other process. Keap1, arepressor protein, binds to Nrf2 and promotes its degradation by theubiquitin proteasome pathway. Keap1 binding and subsequent promotion ofthe ubiquitination is the major regulatory mechanism for Nrf2 controlledgenes. An example of such an agent is 4-Octyl Itaconate.

In one embodiment, the invention concerns up-regulating the sulfonatingcapacity of hair bearing skin, hair follicles, and/or keratinocyte cellsby applying a composition comprising a direct or indirect inhibitor ofKeap1 (either a protein inhibitor (e.g., competitive/non-competitiveinhibiter of Nrf2 binding) or an agent that reduces the transcriptionexpression of Keap1 mRNA) to a hair follicle cell and/or a keratinocytecell. Examples of direct inhibitors are mir-200a, RTA 408, and LH601A.

In one embodiment, the invention concerns up-regulating the sulfonatingcapacity of hair bearing skin, hair follicles, and/or keratinocyte cellsby applying a composition comprising an agent that down-regulates Keap1(Keap1 gene) expression to a hair follicle cell and/or a keratinocytecell.

In one embodiment, the invention concerns up-regulating the sulfonatingcapacity of hair bearing skin, hair follicles, and/or keratinocyte cellsby applying a composition comprising an inducer of the activity of or anagent that increases the activity of p300/CBP to a hair follicle celland/or a keratinocyte cell. Examples of such agents are agents thatincrease intracellular pH. This can involve increasing intercellular pHto an alkaline pH range of 7.4-11.0.

In one embodiment, the invention concerns up-regulating the sulfonatingcapacity of hair bearing skin, hair follicles, and/or keratinocyte cellsby applying a composition comprising an agent that increases the stateof oxidative stress of a cell, wherein the composition is applied to ahair follicle cell and/or a keratinocyte cell. Examples of oxidativestress inducers are agents that increase intracellular pH (alkaline pHrange 7.4-11.0), such an alkaline solution containing a buffer, e.g.,bicarbonate (e.g., sodium bicarbonate).

In one embodiment, the invention concerns a kit having any of thecompositions and/or therapeutic agents disclosed herein configured toup-upregulate the sulfonating capacity of hair bearing skin, hairfollicles, and/or keratinocyte cells. The kit comprises a dispenser,implant, or pill. The composition and/or therapeutic agent is mixed withminoxidil or is packaged in the dispenser, implant, or pill withminoxidil.

In one embodiment, the invention concerns a kit having any of thecompositions and/or therapeutic agents disclosed herein configured toup-upregulate the sulfonating capacity of hair bearing skin, hairfollicles, and/or keratinocyte cells. The kit comprises a firstdispenser, first implant, or first pill. The kit comprises a seconddispenser, second implant, or second pill. The composition and/ortherapeutic agent is packaged in the first dispenser, first implant, orfirst pill. The minoxidil is packaged in the second dispenser, secondimplant, or second pill.

In one embodiment, the invention concerns up-regulating the sulfonatingcapacity of hair bearing skin, hair follicles, and/or keratinocyte cellsby applying a topical solution containing an agonist of the GR nuclearreceptor. In one embodiment, the invention concerns up-regulating thesulfonating capacity of hair bearing skin, hair follicles, and/orkeratinocyte cells by applying a topical solution containing analkalinizing agent or an alkalinizing agent with a penetration enhancerwith an agonist of the GR nuclear receptor. Examples of GR agonistsinclude, but are not limited to, glucocorticoids.

In one embodiment, the invention concerns up-regulating the sulfonatingcapacity of hair bearing skin, hair follicles, and/or keratinocyte cellsby applying a topical solution containing an agonist of the FXR nuclearreceptor. In one embodiment, the invention concerns up-regulating thesulfonating capacity of hair bearing skin, hair follicles, and/orkeratinocyte cells by applying a topical solution containing analkalinizing agent or an alkalinizing agent with a penetration enhancerwith an agonist of the FXR nuclear receptor. Examples of FXR agonistsinclude, but are not limited to, bile acids, GW4064, AGN29, AGN31,cafestol, fexaramine, XL335, WAY-362450, FXR-450, obeticholic acid(OCA), PX 20350, and DY 268.

In one embodiment, the invention concerns up-regulating the sulfonatingcapacity of hair bearing skin, hair follicles, and/or keratinocyte cellsby applying a topical solution containing an agonist of the LXRα nuclearreceptor. In one embodiment, the invention concerns up-regulating thesulfonating capacity of hair bearing skin, hair follicles, and/orkeratinocyte cells by applying a topical solution containing analkalinizing agent or an alkalinizing agent with a penetration enhancerwith an agonist of the LXRα nuclear receptor. Examples of LXRα agonistsinclude, but are not limited to, GW3965, T0901317, paxiline, F3methylAA,and acetylpodocarpic dimer (APD).

In one embodiment, the invention concerns up-regulating the sulfonatingcapacity of hair bearing skin, hair follicles, and/or keratinocyte cellsby applying a topical solution containing an agonist of the VDR nuclearreceptor. In one embodiment, the invention concerns up-regulating thesulfonating capacity of hair bearing skin, hair follicles, and/orkeratinocyte cells by applying a topical solution containing analkalinizing agent or an alkalinizing agent with a penetration enhancerwith an agonist of the VDR nuclear receptor. Examples of VDR agonistsinclude, but are not limited to, 1α,25-Dihydroxyvitamin D3 andlithocholate.

In one embodiment, the invention concerns up-regulating CYP1A1, 1A2,1B1, 2S1, UGT1A1, or UGT1A6 in hair bearing skin, hair follicles, and/orkeratinocyte cells by applying a topical solution containing an agonistof the AhR nuclear receptor. In one embodiment, the invention concernsup-regulating the sulfonating capacity of hair bearing skin, hairfollicles, and/or keratinocyte cells by applying a topical solutioncontaining an alkalinizing agent or an alkalinizing agent with apenetration enhancer with an agent that up-regulates CYP1A1, 1A2,1B1,2S1, UGT1A1, or UGT1A6. The topical solution containing an agonistof the AhR nuclear receptor. Examples of AhR agonists include, but arenot limited to, PAHs, TCDD (other PHAHs), β-naphthoflavone, indigoids,tryptophan metabolites, omeprazole, and lansoprazole.

In one embodiment, the invention concerns up-regulating CYP2A6, 2B6,2C8, 2C9, 2C19, 3A4, UGT1A1, SULT1A1, ALAS, MRP2, or MRP3 concentrationin hair bearing skin, hair follicles, and/or keratinocyte cells byapplying a topical solution containing an agonist of the CAR nuclearreceptor. In one embodiment, the invention concerns up-regulating thesulfonating capacity of hair bearing skin, hair follicles, and/orkeratinocyte cells by applying a topical solution containing analkalinizing agent or an alkalinizing agent with a penetration enhancerwith an agonist of the CAR nuclear receptor. Examples of CAR agonistsinclude, but are not limited to, phenobarbital, phenytoin,carbamazepine, CITCO (human), TCPOBOP (mouse), clotrimazole, and Yin ZhiWuang (many PXR agonists are also CAR agonists, and vice versa).

In one embodiment, the invention concerns up-regulating CYP2B6, 2C8,2C9, 2C19, 3A4, 3A7, 7A1, SULT2A1, UGT1A1, 1A3, 1A4, PAPSS2, ALAS, MDR1,or AhR concentration in hair bearing skin, hair follicles, and/orkeratinocyte cells by applying a topical solution containing an agonistof the PXR nuclear receptor. In one embodiment, the invention concernsup-regulating the sulfonating capacity of hair bearing skin, hairfollicles, and/or keratinocyte cells by applying a topical solutioncontaining an alkalinizing agent or an alkalinizing agent with apenetration enhancer with an agonist of the PXR nuclear receptor.Examples of PXR agonists include, but are not limited to, amprenavir,avasimibe, bosentan, bile acids, carbamazepine, clindamycin,clotrimazole, cortisol, cyproterone acetate, dicloxacillin, efavirenz,etoposide, dexamethasone, genistein, griseofulvin, guggulsterone,guttiferone G, garcinol, Isogarcinol hyperforin (Saint John's Wort),indinavir, lovastatin, mifepristone, nafcillin, nelfinavir, nifedipine,omeprazole, paclitaxel, PCBs, phenobarbital, phthalate monoesters,5β-pregnane-3,20-dione, rifabutin, rifampin, ritonavir, saquinavir,simvastatin, spironolactone, sulfinpyrazole, TAO, tetracycline,topotecan, transnanoclor, troglitazone, verapamil, vitamin E, vitaminK2, artemisinin, PCN, LCA, cafestol, SR-12813, rifaximin, mevastatin,TO901317, Solomonsterol A, and meclizine.

In one embodiment, the invention concerns up-regulating BSEP, I-BABP,MDR3, UGT2B4, SULT2A1, OATP8, PPARα, or SHP concentration in hairbearing skin, hair follicles, and/or keratinocyte cells by applying atopical solution containing an agonist of the FXR nuclear receptor.Examples of FXR agonists include, but are not limited to, bile acids,GW4064, AGN29, and AGN31.

In one embodiment, the invention concerns up-regulating the sulfonatingcapacity of hair bearing skin, hair follicles, and/or keratinocyte cellsby applying a topical solution containing an alkalinizing agent or analkalinizing agent with a penetration enhancer with an agonist of theRXR nuclear receptor. Examples of RXR agonists include but are notlimited to 9-cis-retinoic acid, all trans retinoic acid, ATRA, retinol(or retinal or retinaldehyde), retin-A,(E)-5,8,11,14,17,20-docosahexaenoic acid, lithocholic acid, phytanicacid, 9cUAB30, AGN194204, CD3254L, G100268, LG101305, methoprene acid,PA024, SR11217, SR11237 (BMS649), DEC1, DR5III, PRIC295, bexarotene,CD3254, decosahexaenoic, flurobexarotene, LG 100268, LG 100754,isotretinoin, etc.

In one embodiment, the invention concerns up-regulating the sulfonatingcapacity of hair bearing skin, hair follicles, and/or keratinocyte cellsby applying a topical solution containing an alkalinizing agent or analkalinizing agent with a penetration enhancer with an agonist of theRAR nuclear receptor. Examples of RAR agonists include but are notlimited to all-trans-retinoic acid, ATRA, retinol (or retinal orretinaldehyde), retin-A, 9-cis-retinoic acid, all-trans-5,6-epoxyretinoic acid, DR5III, isotretinoin, AC 261066, AC 55649, adapalene, AM580, AM 80, BMS 753, BMS 961, BMS 453, CD 1530, CD 2314, CD 437, Ch 55,tazarotene, TTNPB, AR-7, FOXO1, SMRT, N-CoR, SMRTER, EC 19, etc.

In one embodiment, the invention concerns up-regulating the sulfonatingcapacity of hair bearing skin, hair follicles, and/or keratinocyte cellsby applying a topical solution containing an alkalinizing agent or analkalinizing agent with a penetration enhancer with an agonist of theFXR nuclear receptor. Examples of FXR agonists include, but are notlimited to, bile acids, GW4064, AGN29, and AGN31.

In one embodiment, the invention concerns increasing the activity ofsulfotransferase by generating an embodiment of the compositiondisclosed herein and incorporating the composition into a solution. Themethod can further include adjusting the pH of the solution to begreater than 7. The method can involve applying the solution with thecomposition to skin of a person. Having a solution with an embodiment ofthe composition, wherein the solution has a pH greater than 7 canincrease the activity of sulfotransferase, which can include increasingthe SULT1A1 enzyme metabolic activity.

Nuclear receptor agonists may be administered to the hair follicle orscalp to increase the sulfonating capacity of hair bearing skin, hairfollicles, and/or keratinocyte cells, treat or prevent alopecia and theother disorders discussed herein. It is specifically contemplated that anuclear receptor agonists known in the art or disclosed herein can beadministered to the hair follicle or the scalp in combination with anagent that retards systemic absorption of the agent across the dermis.In this manner, agents that might otherwise have unwanted systemiceffects can be used to treat, reduce or prevent alopecia or otherdisorders discussed herein while avoiding such systemic side effects.One formulation of agents for topical administration in a manner thatavoids systemic absorption is discussed in detail in U.S. 2009/0068287,which is incorporated herein by reference in its entirety.

In one aspect, the therapeutic agent, such as the nuclear receptoragonist or an alkalinizing agent or an alkalinizing agent with apenetration enhancer, is applied to a skin section, such as a section ofthe scalp, that contains at least one hair follicle.

In one aspect of the present invention, a kit for measuring the pH of asubject's hair follicle is used to diagnose androgenetic alopecia. Inone aspect of the present invention a kit for measuring the pH of asubject's hair follicle is used to diagnose minoxidil response. Inanother aspect a kit for measuring the pH of a subject's hair follicleis used to detect stem cell proliferation in a hair follicle. In yetanother embodiment the efficacy of a hair growing treatment is assessedby measuring the pH of a subject's hair follicle at baseline with a kitfor measuring the pH of a subject's hair follicle, using a hair growingtreatment for a prescribed time, and finally measuring the pH of asubject's hair follicle after using the treatment for comparison tobaseline.

In one aspect of the present invention, a kit for measuring the pH of asubject's hair follicle is used. The kit comprising Bromothymol Blue(0.04%) and a 20× magnification stereo microscope equipped with a 5MPColor Digital Eyepiece Microscope Camera.

In one embodiment of the present invention intracellular alkalization isachieved by activating one or more ion channels that regulate cytoplasmpH such as but not limited to: Na⁺—H⁺ exchangers (NHEs), Na⁺— HCO₃ ⁻co-transporters (NBCs), Na⁺-dependent Cl⁻—HCO₃ ⁻ exchangers (NDCBEs),Na⁺—K⁺-ATPase pumps (NKAs). Activating the ion channels that result inintracellular alkalization can be made by using one or more of thefollowing agents: saxitoxin, neosaxitoxin, tetrodotoxin, oxcarbazepine,carbamazepine, quinidine, procainamide, disopyramide, lidocaine,mexiletine, tocainide, phenytoin, encainide, flecainide, moricizine,propafenone, aconitine, batrachotoxin, robustoxin, versutoxin,ciguatoxins, DDT, pyrethrines, fenvalerate, solnatide (AP301), ambroxol,bromhexine, articaine hydrochloride, articaine, benzamil, bupivacaine,camostat mesylate, carbamazepine, cariporide, 3′,4′-dichlorobenzamil,disopyramide, encainide, flecainide acetate, GMQ, halofantrine,lappaconitine, levobupivacaine, lidocaine, lidocaine hydrochloride,lidocaine N-ethyl chloride, lorcainide, metolazone, mexiletine, ouabainoctahydrate, PF-01247324, PF-04531083, PF-04856264, PF-05089771,PF-06305591, pilsicainide, α-pompilidotoxin, procaine, propafenone,ProTx-II, pyrethrum, quinidine, quinidine sulfate, ralfinamide,ralfinamide mesylate, rostafuroxin, safinamide, safinamide mesylate,tocainide, tolperisone, UCL 2077, veratridine, zoniporide, etc.

In one embodiment of the present invention, intracellular alkalizationis achieved by up-regulating one or more ion channels that regulatecytoplasm pH such as but not limited to: Na⁺—H⁺ exchangers (NHEs),Na⁺—HCO₃ ⁻ co-transporters (NBCs), Na⁺-dependent Cl⁻—HCO₃ ⁻ exchangers(NDCBEs), Na⁺—K⁺-ATPase pumps (NKAs). Up-regulating the ion channelsthat result in intracellular alkalization can be made by using one ormore of the following agents: angiotensin II, catecholamines,endothelin-1, glucocorticoids, NPY, thyroid hormones, etc.

In one embodiment of the present invention, intracellular alkalizationis achieved by inhibiting one or more ion channels that regulatecytoplasm pH such as but not limited to: Cl⁻—HCO₃ ⁻ or anion exchangers(AEs), Ca²⁺-ATPases (PMCAs). Inhibiting the ion channels that result inintracellular alkalization can be made by using one or more of thefollowing agents: cholestyramine, colestipol, colesevelam, rifampicin,naltrexone, naloxone, sertraline, EIPA, acetazolamide, amlodipine(Norvasc), aranidipine (Sapresta), azelnidipine (Calblock), barnidipine(HypoCa), benidipine (Coniel), cilnidipine (Atelec, Cinalong, Siscard),clevidipine (Cleviprex), efonidipine (Landel), felodipine (Plendil),isradipine (DynaCirc, Prescal), lacidipine (Motens, Lacipil),lercanidipine (Zanidip), manidipine (Calslot, Madipine), nicardipine(Cardene, Carden SR), nifedipine (Procardia, Adalat), nilvadipine(Nivadil), nimodipine (Nimotop), nisoldipine (Baymycard, Sular, Syscor),nitrendipine (Cardif, Nitrepin, Baylotensin), pranidipine (Acalas),fendiline, gallopamil, verapamil (Calan, Isoptin), diltiazem (Cardizem),mibefradil, bepridil, flunarizine, fluspirilene, fendiline,gabapentinoids, gabapentin, pregabalin, ziconotide, niflumic acid,anthracene-9-carboxylic acid, etc.

In one embodiment of the present invention, intracellular alkalizationis achieved by down-regulating one or more ion channels that regulatecytoplasm pH such as but not limited to: Cl⁻—HCO₃ ⁻ or anion exchangers(AEs), Ca²⁺-ATPases (PMCAs). Down-regulating the ion channels thatresult in intracellular alkalization can be made by using one or more ofthe following agents: angiotensin II, catecholamines, endothelin-1,glucocorticoids, NPY, thyroid hormones, etc.

In one embodiment of the present invention, intracellular acidificationis achieved by activating one or more ion channels that regulatecytoplasm pH such as but not limited to: Cl⁻—HCO₃ ⁻or anion exchangers(AEs), Ca²⁺-ATPases (PMCAs). Activating the ion channels that result inintracellular acidification can be made by using one or more of thefollowing agents: Bay K8644, nifedipine, ambroxol, lubiprostone, Amitiza(Pro), 1,10-phenanthroline, or GABA-A receptor agonists (e.g lorazepam),etc.

In one embodiment of the present invention, intracellular acidificationis achieved by up-regulating one or more ion channels that regulatecytoplasm pH such as but not limited to: Cl⁻— HCO₃ ⁻ or anion exchangers(AEs), Ca²⁺-ATPases (PMCAs). Up-regulating the ion channels that resultin intracellular acidification can be made by using one or more of thefollowing agents: angiotensin II, catecholamines, endothelin-1,glucocorticoids, NPY, thyroid hormones, etc.

In one embodiment of the present invention, intracellular acidificationis achieved by inhibiting one or more ion channels that regulatecytoplasm pH such as but not limited to: Na⁺—H⁺ exchangers (NHEs),Na⁺—HCO₃ ⁻ co-transporters (NBCs), Na⁺-dependent Cl⁻—HCO₃ ⁻ exchangers(NDCBEs), Na⁺—K⁺ ATPase pumps (NKAs). Inhibiting the ion channels thatresult in intracellular acidification can be made by using one or moreof the following agents: S-(N-ethyl-N-isopropyl) amiloride, zoniporide,cariporide, KR-32568[5-(2-Methyl-5-fluorophenyl)furan-2-ylcarbonyl]guanidine, eniporide,EMD87580 [(2-methyl-4,5-di-(methylsulfonyl)-benzoyl)-guanidine], HMA[5-(N,N-hexamethylene)-amiloride], KR-33028 (4-cyano(benzo[b]thiophene-2-carbonyl)guanidine), S0859,[2-chloro-N[[2′-[(cyanoamino)sulfonyl][1,1′-biphenyl]-4-yl]methyl]-N-[(4-methylphenyl)methyl]-benzamide,levetiracetam, hydrochlorothiazide (HCTZ), ouabain, dihydroouabain,lanatoside C, bufalin, digitoxin, digoxin, strophantidin, ouabagenin,etc.

In one embodiment of the present invention, intracellular acidificationis achieved by down-regulating one or more ion channels that regulatecytoplasm pH such as but not limited to: Na⁺—H⁺ exchangers (NHEs),Na⁺—HCO₃ ⁻ co-transporters (NBCs), Na⁺-dependent Cl⁻—HCO₃ ⁻ exchangers(NDCBEs), Na⁺—K⁺-ATPase pumps (NKAs). Down-regulating the ion channelsthat result in intracellular acidification can be made by using one ormore of the following agents: angiotensin II, catecholamines,endothelin-1, glucocorticoids, NPY, thyroid hormones, etc.

In one embodiment of the present invention, intracellular alkalizationor acidification is achieved by activating endogenous pH sensors such asbut not limited to acid-sensing ion channels, pH-sensing ionotropicreceptors, pH-sensing metabotropic receptors, transient receptorpotential ion channels, TRPV1, TRPC4, TRPC5, TRPP2, and purinoceptors,etc.

Formulations

The therapeutic agents, particularly the nuclear receptor agonistsand/or the alkalinizing agents, described herein and used in the presentmethods may be formulated into compositions according to the knowledgeof one of skill in the art. In one embodiment, the nuclear receptoragonist and/or the alkalinizing agent or other inducer ofsulfotransferase is formulated for topical slow or prolonged release. Asbut one example, in one embodiment the inducer of sulfotransferase isencapsulated for slow release.

The therapeutic agents, particularly the nuclear receptor agonistsand/or the alkalinizing agent, described herein and used in the presentmethods may be formulated into compositions according to the knowledgeof one of skill in the art. In one embodiment, the nuclear receptoragonist or other inducer of sulfotransferase is encapsulated in order toincrease the water solubility of the therapeutic agent. In anotherembodiment, the nuclear receptor agonist or other inducer ofsulfotransferase is encapsulated in order to reduce the loss throughdegradation of therapeutic agent, for example, to reduce oxidation ofthe therapeutic agent.

In one embodiment hyperforin is encapsulated to overcome its poor watersolubility and facile oxidative degradation.

In one embodiment of the present invention a diagnostic test is used todetermine if a subject will likely have sulfotransferase up-regulated bya particular PXR agonist. For example, a genetic test of the PXR genemay identify if human PXR was rendered hyperforin insensitive viamutagenesis of Leu308 to phenylalanine. Other methods are possible.

In one embodiment of the present invention a PXR agonist, CAR agonist,an alkalinizing agent, and/or acidifying agent is secreted from agenetically modified organism (GMO) that is transplanted to the humanskin.

In one embodiment of the present invention a PXR agonist, CAR agonist,and/or an alkalinizing agent is secreted from a bacterial organism thatis applied as a pro-biotic to the human skin

In one embodiment of the present invention the use of a topicalcomposition applied to the scalp that up-regulates the sulfonatingcapacity of the hair follicle is used to increase the efficacy of alow-dose oral minoxidil. The method further includes the use of topicalcomposition applied to the scalp that up-regulates the sulfonatingcapacity of the hair follicle to target minoxidil activation to thescalp for a subject receiving oral, low-dose minoxidil. Examples of orallow-dose minoxidil include dosage less than 0.5 mg once daily. Otherexamples include 0.45 mg, 0.4 mg, 0.35 mg, 0.30 mg, 0.25 mg, 0.20 mg,0.15 mg, 0.10 mg, 0.05 mg, 250 μM, 10 μM, used once daily.

In another embodiment, the nuclear receptor agonist or other inducer ofsulfotransferase or PAPS is formulated in a shampoo, a foam, ointment,spray, solution, gel, slow release capsule, oral tablet, dry shampoo, orany similar compound or delivery vehicle or methodology. Topicalapplication is preferred. In one embodiment, the composition isformulated in a topical cream. In another embodiment, the composition isformulated in a hair styling product selected from the group consistingof a styling gel, a styling foam, and a hair conditioner.

In another embodiment, the composition may comprise an exfoliating agentto promote abrasion of the surface of the scalp. Examples of theexfoliating agent include (1) inorganic and/or metallic particles suchas: boron nitride, in body-centered cubic form (Borazon®);aluminosilicate (e.g. nepheline); zircon; mixed oxides of aluminum suchas emery; zinc oxide; aluminum oxides such as aluminas or corundum;titanium oxide; titanium oxide coated mica; carbides, in particularsilicon carbide (carborundum); or other metal oxides; metals, and metalalloys such as iron shot, steel shot, and in particular perlite;silicates such as glass, quartz, sand, or vermiculite; calcium carbonate(e.g. Bora-Bora sand or Rose de Brignoles sand) or magnesium carbonate;sodium chloride; pumice stone; amorphous silica; diamond; ceramics, and(2) organic particles such as: fruit stones, in particular apricotstones, e.g. Scrubami® apricot; wood cellulose, e.g. ground bamboo stem;coconut shell, e.g. coconut exfoliator; polyamides, in particularNylon-6; sugars; plastic microbeads, e.g. polyethylenes orpolypropylenes; ground walnut; ground apricot seed; ground shells, and(3) mixed particles associating organic and inorganic compounds, andparticles coated in the above compounds. The exfoliating agents may bein the form of microbeads of less than five millimeters in its largestdimension that have an exfoliating effect.

In another embodiment, the composition may comprise an exfoliating agentto promote absorption of the nuclear receptor agonist and/or thealkalinizing agent into scalp. An example of the exfoliating agentinclude salicylic acid.

In one embodiment, the composition comprising a nuclear receptor agonistand/or the alkalinizing agent can be formulated as a drug (which mayinclude a drug with minoxidil). In one embodiment, the compositioncomprising a nuclear receptor agonist and/or an alkalinizing agent canbe formulated as a cosmetic product. In one embodiment, the compositioncomprising a nuclear receptor agonist and/or the alkalinizing agent canbe formulated as a cosmetic product to be used before using minoxodil.

The amount of therapeutic agent present in the composition may bedetermined by one of skill in the art using known methodologies. Incertain embodiments, the nuclear receptor agonist and/or thealkalinizing agent or other inducer of sulfotransferase or PAPS ispresent in the composition in a concentration from about 0.0020% to0.0030%, or about 0.0025% by weight. In another embodiment, thetherapeutic agent such as a nuclear receptor agonist and/or analkalinizing agent is present in the composition in a concentration ofabout 0.0025%, 0.0033%, 0.005%, 0.01%, 0.02%, 0.025%, or 0.10% byweight.

In other embodiments, the therapeutic agent, such as the nuclearreceptor agonist and/or the alkalinizing agent, is present in thetopical composition for use in the methods disclosed herein in aconcentration from about 0.1% to 35%, about 1.0% to 30%, about 0.2% to30%, about 0.2% to 25%, about 0.2% to 20%, about 0.2% to 15%, about 0.2%to 10%, about 0.2% to 5%, about 0.2% to 4%, about 0.2% to 3%, about 0.2%to 2%, about 0.2% to 1%, about 10.0% to 30%, about 15.0% to 30%, about20.0% to 30%, about 10% to 20%, about 10% to 15%, about 15% to 20%,about 15% to 60%, about 20% to 60%, about 50% to 60%, and about 45% to55% by weight.

In one embodiment, the composition comprises a nuclear receptor agonistand/or an alkalinizing agent in a concentration of about 0.025%, about0.033%, about 0.05%, about 0.1%, about 0.2%, about 0.25%, about 0.30%,about 0.40%, about 1.0%, about 1.5%, about 2.0%, or about 2.5% byweight.

The compositions used in the present disclosure, particularlycompositions containing a nuclear receptor agonist and/or analkalinizing agent, may be formulated with a preservative such as EDTA(0.1-0.5% by weight of the formulation) and/or sodium metabisulfite(0.1-0.5% by weight of the formulation). In some embodiments, thepenetration enhancer is selected from one or more of the groupconsisting of alcohols, glycols, fatty acids, fatty esters, fattyethers, occlusive agents, surface active agents, dimethylaminopropionicacid derivatives, terpenes, sulfoxides, cyclic ethers, amides, andamines. Other components of the formulations used herein may be chosenfrom cosmetically approved excipients known in the art, including water,thickeners, etc.

The composition may be packaged in a kit with an applicator forapplication to the skin. The invention is also directed to a kitcomprising a composition of the therapeutic agent, such as a nuclearreceptor agonist, and an applicator, and to a kit comprising acomposition of the therapeutic agent, such as a nuclear receptoragonist, and a hair brush or comb, particularly a brush or comb thatprovides exfoliating effect on the scalp such that there is lightabrasion after its use that enhances penetration of the therapeuticagent to the AP muscle and/or hair follicle. In one embodiment, thetherapeutic agent is provided in a metered dose applicator that providesfor a fixed volume of the composition to be administered with eachadministration, such as 1 ml of the topical composition peradministration.

The composition may be packaged in a kit including a topical minoxidilformulation. For example, a 2% minoxidil topical solution, a 3% topicalminoxidil solution, a 5% topical minoxidil solution, a 5% topicalminoxidil foam, a 10% topical minoxidil solution

It will be understood that the ranges described above, and throughoutthis document, are also intended to encompass single values containedwithin these ranges. For example, for a formulation comprising aparticular ingredient in a range between 1-50%, a percentage of 5% or49% is also intended to be disclosed.

Therapeutic Agents

The methods of the present disclosure may be used with a nuclearreceptor agonist or other compound that causes induction ofsulfotransferases or PAPSS. Suitable nuclear receptor agonists can beutilized including but are not limited to, amprenavir, avasimibe,bosentan, bile acids, carbamazepine, clindamycin, clotrimazole,cortisol, cyproterone acetate, dicloxacillin, efavirenz, etoposide,dexamethasone, genistein, griseofulvin, guggulsterone, hyperforin (SaintJohn's Wort), indinavir, lovastatin, mifepristone, nafcillin,naringenin, nelfinavir, nifedipine, omeprazole, paclitaxel, PCBs,phenobarbital, phthalate monoesters, 5β-pregnane-3,20-dione, quercetin,rifabutin, rifampin, ritonavir, saquinavir, simvastatin, spironolactone,sulfinpyrazole, TAO, tetracycline, topotecan, transnanoclor,troglitazone, verapamil, vitamin E, vitamin K2, artemisinin, PCN, LCA,cafestol, SR-12813, rifaximin, mevastatin, TO901317, Solomonsterol A,meclizine, fibrates, WY-14,643, perfluorodecanoic acid, bile acids,GW4064, AGN29, AGN31, cafestol, fexaramine, XL335, WAY-362450, FXR-450,obeticholic acid (OCA), PX 20350, and DY 268. Additionally, derivativesof nuclear receptor agonists can be utilized including derivatives ofthe compounds mentioned above. In other embodiments, a prodrug that isactivated to become a nuclear receptor agonist can be utilized.

In one embodiment, the nuclear receptor agonist is hyperforin, or apharmaceutically acceptable salt or hydrate thereof, in a composition ina concentration of 0.0025% to 40%, 0.0025% to 25% by weight, or 0.005%to 22.5% by weight, or 0.0075% to 20% by weight, or 1% to 17.5% byweight, or 1.5% to 15% by weight, or 2% to 14.5% by weight, or 2.5% to14% by weight, or 5% to 13.5% by weight, or 7.5% to 12.5% by weight, or8% to 12% by weight, or 8.5% to 11.5% by weight, or 9% to 11% by weight,or 9.25% to 10.75% by weight, or 9.5% to 10.5% by weight, or 9.6% to10.4% by weight, or 9.7% to 10.3% by weight, or 9.8% to 10.2% by weight,or 9.9% to 10.1% by weight, or 9.95% to 10.05% by weight, or 9.96% to10.04% by weight, or 9.97% to 10.03% by weight, or 9.98% to 10.02% byweight, or 9.99% to 10.01% by weight.

In one embodiment, the nuclear receptor agonist is a Saint John's Wortextract containing hyperforin, or a pharmaceutically acceptable salt orhydrate thereof, in a composition in a concentration of 0.0025% to 40%,0.0025% to 25% by weight, or 0.005% to 22.5% by weight, or 0.0075% to20% by weight, or 1% to 17.5% by weight, or 1.5% to 15% by weight, or 2%to 14.5% by weight, or 2.5% to 14% by weight, or 5% to 13.5% by weight,or 7.5% to 12.5% by weight, or 8% to 12% by weight, or 8.5% to 11.5% byweight, or 9% to 11% by weight, or 9.25% to 10.75% by weight, or 9.5% to10.5% by weight, or 9.6% to 10.4% by weight, or 9.7% to 10.3% by weight,or 9.8% to 10.2% by weight, or 9.9% to 10.1% by weight, or 9.95% to10.05% by weight, or 9.96% to 10.04% by weight, or 9.97% to 10.03% byweight, or 9.98% to 10.02% by weight, or 9.99% to 10.01% by weight.

In one embodiment, the nuclear receptor agonist is vitamin K2, or apharmaceutically acceptable salt or hydrate thereof, in a composition ina concentration of 0.0025% to 40%, 0.0025% to 25% by weight, or 0.005%to 22.5% by weight, or 0.0075% to 20% by weight, or 1% to 17.5% byweight, or 1.5% to 15% by weight, or 2% to 14.5% by weight, or 2.5% to14% by weight, or 5% to 13.5% by weight, or 7.5% to 12.5% by weight, or8% to 12% by weight, or 8.5% to 11.5% by weight, or 9% to 11% by weight,or 9.25% to 10.75% by weight, or 9.5% to 10.5% by weight, or 9.6% to10.4% by weight, or 9.7% to 10.3% by weight, or 9.8% to 10.2% by weight,or 9.9% to 10.1% by weight, or 9.95% to 10.05% by weight, or 9.96% to10.04% by weight, or 9.97% to 10.03% by weight, or 9.98% to 10.02% byweight, or 9.99% to 10.01% by weight.

In one embodiment, the Nuclear receptor agonist is genistein, or apharmaceutically acceptable salt or hydrate thereof, in a composition ina concentration at a range of 0.25%, 0.5%, 0.75%, 1%, 1.5%, 2%, 2.5%,5%, 7.5%, 8%, 8.5%, 9%, 9.25%, 9.5%, 9.6%, 9.7%, 9.8%, 9.9%, 9.95%,9.96%, 9.97%, 9.98%, or 9.99% by weight as the lower weight limit of therange to an upper weight limit of 10.01%, 10.02%, 10.03%, 10.04%,10.05%, 10.1%, 10.2%, 10.3%, 10.4%, 10.5%, 10.75%, 11%, 11.5%, 12%,12.5%, 13.5%, 14%, 14.5%, 15%, 17.5%, 20%, 22.5%, 25%, 30%, 35%, 40%,45%, or 50% by weight (e.g., a range of 0.25% to 10.01%, 0.25% to10.02%, 0.5% to 10.01%, 0.5% to 10.02%, etc.).

In some embodiments, provided herein is a nuclear receptor agonistformulated with a carrier or delivery vehicle optimized for delivery ofthe nuclear receptor agonist to the scalp. A nuclear receptor agonistcan be released using several different formulations or release methodsincluding time release, creams, ointments, sprays, capsules, or otherrelease methods. For instance the nuclear receptor agonist can beincorporated into a shampoo for utilization during showering. In otherembodiments, the nuclear receptor agonist can be included in ointmentsor other topical creams that could be applied to the scalp so that itcan be slowly absorbed into the skin. In other embodiments, the Nuclearreceptor agonist can be included in a liquid spray or aerosol medium tobe applied to the scalp. In other embodiments, the nuclear receptoragonist can be incorporated into capsules or other slow release vehiclesthat would allow the chemical or agent to be slowly released into thedermis of the scalp. Capsules or vehicles that encapsulate the nuclearreceptor agonist can include, but are not limited to, liposomes,non-ionic liposomes, niosomes, novasome I, erythromycin-Zn complex,microspheres, nanoparticles, solid lipid nanoparticles, andnanoemulsions. In some embodiments, this can include a gel or foam thatis applied to the scalp. It is specifically contemplated that thenuclear receptor agonist can be formulated in hair care products such asa shampoo, styling gel, styling foam, hair conditioner, hair serum, ahair mask, etc.

In some embodiments, provided herein is alkalinizing agent or analkalinizing agent with a penetration enhancer formulated with a carrieror delivery vehicle optimized for delivery of the alkalinizing agent tothe scalp. An alkalinizing agent can be released using several differentformulations or release methods including time release, creams,solutions, lotions, serums, ointments, sprays, capsules, or otherrelease methods. For instance the alkalinizing agent can be incorporatedinto a shampoo for utilization during showering. In other embodiments,the alkalinizing agent can be included in ointments or other topicalcreams that could be applied to the scalp so that it can be slowlyabsorbed into the skin. In other embodiments, the alkalinizing agent canbe included in a liquid spray or aerosol medium to be applied to thescalp. In other embodiments, the alkalinizing agent can be incorporatedinto capsules or other slow release vehicles that would allow thechemical or agent to be slowly released into the dermis of the scalp.Capsules or vehicles that encapsulate the alkalinizing agent or analkalinizing agent with a penetration enhancer can include, but are notlimited to, liposomes, non-ionic liposomes, niosomes, novasome I,erythromycin-Zn complex, microspheres, nanoparticles, solid lipidnanoparticles, and nanoemulsions. In some embodiments, this can includea gel or foam that is applied to the scalp. It is specificallycontemplated that the alkalinizing agent or an alkalinizing agent with apenetration enhancer can be formulated in hair care products such as ashampoo, styling gel, styling foam, hair conditioner, hair serum, a hairmask, etc.

In some embodiments, provided herein is acidifying agent or anacidifying agent with a penetration enhancer formulated with a carrieror delivery vehicle optimized for delivery of the acidifying agent tothe skin. An acidifying agent can be released using several differentformulations or release methods including time release, creams,ointments, sprays, capsules, solutions, deodorants (solid or liquid),antiperspirants (solid or liquid), moisturizers, shaving creams, shavinggels, lotions or other release methods. For instance the acidifyingagent can be incorporated into an antiperspirant to use under the arms.In other embodiments, the acidifying agent can be included inmoisturizer lotions or other topical creams that could be applied to thelegs so that it can be slowly absorbed into the skin. In otherembodiments, the acidifying agent can be included in a shaving gel thatcan be applied into the beard. In other embodiments, the acidifyingagent can be incorporated into capsules or other slow release vehiclesthat would allow the chemical or agent to be slowly released into thedermis of the skin. Capsules or vehicles that encapsulate the acidifyingagent or an acidifying agent with a penetration enhancer can include,but are not limited to, liposomes, non-ionic liposomes, niosomes,novasome I, erythromycin-Zn complex, microspheres, nanoparticles, solidlipid nanoparticles, and nanoemulsions. In some embodiments, this caninclude a gel or foam that is applied to the skin. It is specificallycontemplated that the acidifying agent or an acidifying agent with apenetration enhancer can be formulated in skin care products such as amoisturizer lotion, deodorant, antiperspirant, moisturizers, shavingcreams, shaving gels, etc.

Any of the aforementioned formulations can be used routinely, e.g., oncedaily, twice daily, every other day, once a week. Routine use of thenuclear receptor agonist, alkalinizing agent, and/or an alkalinizingagent, with or without a penetration enhancer, would be indicated as anadjuvant therapy for minoxidil in androgenetic alopecia patients. In isspecifically envisioned that a composition (e.g., a shampoo) of any ofthe aforementioned nuclear receptor agonists, alkalinizing agent, and/oran alkalinizing agent can be used daily by a person using minoxidil toincrease the effectiveness of minoxidil.

In cases where encapsulation techniques have been implemented in theabove embodied formulations—for example, liposomes, micelles, nanogels,lipid nanoparticles, selenium nanoparticles, bilosomes, dendrimersand/or carbon nanotubes—the stability of the formula will requireoptimization to prevent degradation. For example, liposomes are vesiclestructures created from one (unilamellar) or more bilayers ofamphipathic lipids. In such systems lipids are often in direct contactwith aqueous solution and are subject to hydrolysis (the breakdown oflipids from water moisture) and oxidation (degradation of lipids due tothe loss of electrons). Similarly, delivery systems using encapsulationtechniques are optimized for stratum corneum penetration via the size ofthe encapsulated structure. For example, structural instabilities suchas aggregation or the conversion of unilamellar structures tomultilamellar or other liposome structures in solution would decreasethe efficacy of payload delivery through skin.

Many measurement techniques have been described to track the degradationof encapsulated structures for formula stability optimization. Forexample, subsequent to hydrolysis, free fatty acids are produced and canbe detected with UV-visible (UV/Vis) spectroscopy. UV/Vis spectroscopycan also be used to detect peroxidized lipids if oxidative degradationhas occurred in a formula. Vesicle structural instabilities can bemonitored by probing the hydrodynamic radius of liposomes with thetechniques of dynamic light scattering, cryogenic transmission electronmicroscopy, and small-angle neutron scattering.

Formulas utilizing encapsulation techniques to increase dermalpenetration can be optimized by adjusting pH, temperature, and particlesize (extrusion). In one embodiment of the present invention, aliposomal solution is stabilized by adjusting the pH to a range of 7-11.In another embodiment the pH is adjusted to a range of 7.5 to 10. In yetanother embodiment, a liposomal solution is stabilized by adjusting thepH to a range of 8.0-8.7. In one embodiment a buffering solution, forexample, acetate, citrate, or HEPES is used to stabilize the pH of aliposomal solution. In another embodiment, a Tris buffer is used. Inanother embodiment, a carbonate buffering or a phosphate bufferingsystem is used. In one embodiment of the present invention, a liposomalsolution is stabilized by adjusting the pH to a range of 8.0-9.0 using aTris buffer.

In one embodiment of the present invention a buffering system forincreasing the shelf-life of a liposomal solution is optimized usingUV-visible (UV/Vis) spectroscopy measurements over time. In yet anotherembodiment, samples of optimized liposomal solutions are stored atelevated temperatures to accelerate degradation prior to UV-visible(UV/Vis) spectroscopy measurements over time.

In one embodiment of the present invention, oxidative degradation of aliposomal solution is prevented by the addition of an antioxidant. Inone embodiment, oxidative degradation of a liposomal solution isprevented by the addition of ascorbic acid, monothioglycerol, potassiummetabisulfite, sodium bisulfite, sodium formaldehyde sulfoxylate, sodiumsulfite, sodium thiosulfate, tocopherol, sodium metabisulfite, butylatedhydroxytoluene (BHT), butylated hydroxyanisole (BHA), ascorbylpalmitate, or propyl gallate. In one embodiment, oxidative degradationof a liposomal solution is prevented by the addition of acai oil, alphalipoic acid, green tea extract, retinol, vitamin C, coenzyme Q10 (CoQ-10), isoflavones, polyphenols, curcumin, turmeric, pomegranate,rosemary extract, glutathione, selenium, or zinc. In yet anotherembodiment of the present invention a chelating molecule is In oneembodiment ethylenediaminetetraacetic acid (EDTA), disodium EDTA,tetrasodium EDTA, pentasodium penetate, sodium metasilicate andphosphate derivatives, etidronic acid and its derivatives, or galactaricacid are added to capture free radicals in solution and preventoxidation of lipids in a liposomal containing formula.

In one embodiment of the present invention, a pH range is chosen suchthat it is optimal for preventing degradation of a liposomal solutionand simultaneously within a range of pHs optimal for inducing theupregulation of SULT1A1. In another embodiment of the present invention,a pH range of 8.0-9.0 is used to both stabilize a liposomal solution andinduce the expression of SULT1A1. In one embodiment of the presentinvention, a stable liposomal solution of encapsulated Tris buffer isused to induce the expression of SULT1A1 in the outer root sheath ofhair follicles. In another embodiment, a stable liposomal solution ofencapsulated Tris buffer is prepared using Phosphatidylcholine(Plospholipon 90G). In another embodiment, a stable liposomal solutionof encapsulated Tris buffer is prepared using Phosphatidylcholine(Plospholipon 90G) in an amount between 1 and 15% (w/w). In yet anotherembodiment, a stable liposomal solution of encapsulated Tris buffer isprepared using 2% (w/w) Phosphatidylcholine (Plospholipon 90G). In oneembodiment of the present invention, a liposomal solution containingPhosphatidylcholine (Plospholipon 90G) is protected from hydrolysis andoxidation by adding a Tris buffer at a pH range of 8.0-8.7. In yetanother embodiment of the present invention, a liposomal solutioncontaining Phosphatidylcholine (Plospholipon 90G) is stabilized using aTris buffer at a concentration of 100-500 mM. In another embodiment, aTris buffer at a concentration of 500 mM-1 M is used. In anotherembodiment of the present invention, a liposomal solution containingPhosphatidylcholine (Plospholipon 90G) is stabilized using a Tris bufferat a concentration of 250 mM or 3% (w/w). In another embodiment, aliposomal solution containing Phosphatidylcholine (Plospholipon 90G) and250 mM or 3% (w/w) Tris is used to induce the expression of SULT1A1 inthe outer root sheath of hair follicles.

In one embodiment, phosphatidylcholine (trade name: Phospholipon 90G) isused to encapsulate sodium bicarbonate (pH 10) or TRIS (pH 8). Use ofsodium bicarbonate and/or TRIS up-regulate SULT1A1 in vivo.

Use of RXR, RAR, and Other NR Agonists

Embodiments of the invention can involve use of retinoid X receptor(RXR) agonists, retinoic acid receptor (RAR) agonists, and/or an agonistof another nuclear receptor (NR) in an RXR-NR heterodimer. For example,any one or combination of RXR agonists, RAR agonists, and/or an agonistof another NR in an RXR-NR heterodimer can be used to treat or preventhair loss (e.g., forms of alopecia), increase or improve hair growth,induce sulfotransferase, increase the efficacy of minoxidil, and/orconvert androgenetic alopecia patients who are non-responders tominoxidil into responders. This can be achieved by using any one orcombination of RXR agonists, RAR agonists, and/or an agonist of anotherNR in an RXR-NR heterodimer in accordance with any of the methodsdisclosed herein. As a non-limiting example, embodiments can involve useof an RXR agonist, a RAR agonist, and/or a RXR agonist with an agonistof another NR in an RXR-NR heterodimer to induce the expression ofSULT1A1.

The retinoid X receptor (RXR) is an essential member of thesteroid/thyroid hormone superfamily of nuclear receptors (NRs) thatpredominately function as transcription factors. The natural ligand ofRXR was first proposed to be 9-cis-retinoic acid (9-cis-RA). However,many groups have been unable to detect endogenous 9-cis-RA in cells. Alltrans-retinoic acid (ATRA) had been discovered to be a ligand of RXR.Polyunsaturated fatty acids (PUFAs), such as docosahexaenoic acid (DHA)and a saturated metabolite of chlorophyll, phytanic acid are alsoidentified as RXR ligands.

In the nucleus, RXR functions as a transcription factor. It binds tospecific six-base-pair sequences of DNA in the promoter regions ofgenes. RXR functions as a dimer with either itself (homodimer) oranother NR (heterodimer). Binding by the ligand of the NR partnerdefines the promoter site response element (RE) composed of two sixbase-pair sequences (half-sites) separated by a discrete number of basesto which the RXR-NR heterodimer binds.

Transcriptional activation by RXR-NR dimers can be classified into threecategories: nonpermissive, permissive, and conditionally permissiveheterodimers of RXR and another nuclear receptor (NR).

Examples of transcriptional activation by a nonpermissive heterodimericpartner include thyroid hormone receptor (TR) or vitamin D receptor(VDR). The nonpermissive NR is dominant so that binding by its agonistcontrols the transcriptional complex to initiate gene transcription fromthe nonpermissive-ligand responsive gene transcriptional start site.Binding of an RXR agonist would not enhance the response induced by thebound NR agonist.

Examples of transcriptional activation by a permissive RXR heterodimericpartner include pregnane x receptor (PXR), constitutive androstanereceptor (CAR), farnesoid (bile acid) X receptor (FXR), liver(oxysterol) X receptor (LXR), and peroxisome proliferator-activatedreceptor (PPAR). An agonist of either partner in the heterodimeric pairsuch as RXR-PPAR could bind its own NR initiate gene transcription.Binding of an agonist to the second NR in the dimer would enhance thetranscriptional response induced by first NR—agonist complex eitheradditively or synergistically.

Examples of transcriptional activation by the conditionally permissiveheterodimeric partner can include retinoic acid receptor (RAR). Bindingof the RAR agonist would control the transcriptional response and alsopermit the binding of an RXR agonist. Thus, the RAR—agonist complexwould be permissive. The RXR—agonist complex would then enhance thetranscriptional response induced by the RAR agonist.

As such, a ligand of either RXR or a NR may be used to initiatetranscription of genes. Additionally, in the case of permissive andconditionally permissive transactivation, ligands of RXR or the NR canact synergistically to activate transcription.

In one embodiment of the present invention, a RXR agonist is used toinduce the expression of SULT1A1. Examples of RXR agonists include butare not limited to 9-cis-retinoic acid, all trans retinoic acid, ATRA,retinol (or retinal or retinaldehyde), retin-A,(E)-5,8,11,14,17,20-docosahexaenoic acid, lithocholic acid, phytanicacid, 9cUAB30, AGN194204, CD3254L, G100268, LG101305, methoprene acid,PA024, SR11217, SR11237 (BMS649), DEC1, DR5III, PRIC295, bexarotene,CD3254, decosahexaenoic, flurobexarotene, LG 100268, LG 100754,isotretinoin, etc.

In another embodiment of the present invention, a RAR agonist is used toinduce the expression of SULT1A1. Examples of RAR agonists include butare not limited to all-trans-retinoic acid, ATRA, retinol (or retinal orretinaldehyde), retin-A, 9-cis-retinoic acid, all-trans-5,6-epoxyretinoic acid, DR5III, isotretinoin, AC 261066, AC 55649, adapalene, AM580, AM 80, BMS 753, BMS 961, BMS 453, CD 1530, CD 2314, CD 437, Ch 55,tazarotene, TTNPB, AR-7, FOXO1, SMRT, N-CoR, SMRTER, EC 19, etc.

In yet another embodiment of the present invention, a compound that maybe metabolized by enzymes in the skin to become a RAR or RXR agonist isused to induce the expression of SULT1A1. Examples of a compound thatwill be metabolized to become agonists include but are not limitedretinyl propionate, retinyl palmitate, or retinyl acetate.

In yet another embodiment of the present invention, a RXR agonist isused with an agonist of another NR in an RXR-NR heterodimer to inducethe expression of SULT1A1. Examples of other NRs include thyroid hormonereceptor (TR), vitamin D receptor (VDR), pregnane x receptor (PXR),constitutive androstane receptor (CAR), farnesoid (bile acid) X receptor(FXR), liver (oxysterol) X receptor (LXR), peroxisomeproliferator-activated receptor (PPAR), retinoic acid receptor (RAR),aryl hydrocarbon receptor (AhR), Nrf2, GR, etc.

In one embodiment, the invention concerns up-regulating the sulfonatingcapacity of hair bearing skin, hair follicles, and/or keratinocyte cellsby applying a topical solution containing a salt of minoxidil such thatthe one ion of the salt is an alkalinizing agent that will raise theintracellular pH of cells in the outer root sheath of the hair follicleand the counter ion is minoxidil. Examples of alkalinizing slats ofminoxidil include, but are not limited to, minoxidil bicarbonate,minoxidil citrate, minoxidil carbonate, minoxidil lactate, and minoxidilacetate. In one embodiment the salt is formulated with a penetrationenhancer.

The therapeutic agents, particularly an alkalinizing agent, describedherein and used in the present methods may be formulated intocompositions according to the knowledge of one of skill in the art. Inone embodiment, the alkalinizing agents or other inducer ofsulfotransferase is encapsulated in order to increase the watersolubility of the therapeutic agent. In one embodiment, the alkalinizingagents or other inducer of sulfotransferase is encapsulated in order todecrease the water solubility of the therapeutic agent. In anotherembodiment, the alkalinizing agents or other inducer of sulfotransferaseis encapsulated in order to reduce the loss through degradation oftherapeutic agent, for example, to reduce oxidation of the therapeuticagent. In another embodiment, the alkalinizing agents or other inducerof sulfotransferase is encapsulated in order to promote penetrationthrough the stratum corneum. In some embodiments the alkalinizing agentis placed in a liposome containing lecithin.

Efficacy of treatment to treat or prevent androgenetic alopecia can bedetermined by monitoring the density of hairs on a given area of thesubject's body, e.g., a given area of the scalp. If the rate of hairloss is reduced, e.g., by 10% or more following treatment, the treatmentis effective for the prevention of androgenetic alopecia. Similarly, ifhair density remains the same, the treatment is effective for theprevention of androgenetic alopecia. If the density of hair increases,e.g., by 5% or more, e.g., by 10% or more following treatment, thetreatment is also considered effective for the treatment and/orprevention of androgenetic alopecia.

Efficacy of treatment to treat or prevent androgenetic alopecia can bedetermined by monitoring global photography. For example, the patient oran expert can assess the treatment response utilizing before and afterglobal photographs.

As noted above, it is contemplated that all forms of alopecia canbenefit from the technology described herein. For example, thetechnology described herein can be applicable to prevent or treatandrogenic alopecia.

Any of the activators, inducers, and/or inhibitors disclosed herein canbe formulated as an agent (e.g., a therapeutic agent) to be used as anembodiment of a composition for implementing an embodiment of atreatment disclosed herein.

Any of the compositions, activators, inducers, inhibitors, and/or agentsdisclosed herein can be configured to be administered topically in theform of a shampoo, solution, foam, lotion, gel, spray, or gas. Inaddition, or in the alternative, any of the compositions and/or agentsdiscloses herein can be administered orally, sublingually, by injection,as an implant, or by using a bacteria that secrets an embodiment of theagent.

Embodiments of the treatments disclosed herein can involveadministration of an embodiment of the compositions, activators,inducers, inhibitors, and/or agents disclosed herein in any frequency(e.g., once daily, twice daily, every other day, weekly, monthly, etc.).

Embodiments of any of the compositions or agents disclosed herein can beapplied at a predetermined frequency (e.g., once or twice per day, onceper 24 hours, once per 12 hours, once per 6 hours, etc.), administeredorally, injected (e.g., injected as a slow release formulation), orprovided as an implant.

In one embodiment of the present invention an embodiment of thecomposition or therapeutic agent (e.g., any of the agents or compoundsdisclosed herein) that up-regulates the sulfonating capacity of hairbearing skin is delivered as a slow releasing injectable or implant.Examples of types of delivery systems include but are not limited tomicroparticle-based depot formulations, nanoparticle-based depotformulations, transdermal systems, or implants.

In one embodiment of the present invention an embodiment of thecomposition or therapeutic agent (e.g., any of the agents or compoundsdisclosed herein) that up-regulates the sulfonating capacity of hairbearing skin is delivered as a microparticle-based depot formulation.The formulation can include a polymeric material (e.g., biodegradable)that allows for protection of the drug cargo and control over drugrelease. Examples of polymer choices include but are not limited topoly(lactic-co-glycolic) acid (PLGA), poly(lactic acid) (PLA) andpolyglycolic acid (PGA).

In one embodiment of the present invention an embodiment of thecomposition or therapeutic agent (e.g., any of the agents or compoundsdisclosed herein) that up-regulates the sulfonating capacity of hairbearing skin is delivered as a nanoparticle-based depot formulation. Thenanoparticle-based depot formulation may contain polymericnanoparticles, made from biocompatible and biodegradable materials,gelatin, albumin, synthetic polymers (polylactides,polyalkylcyanoacrylates), or liposomes. Nanoparticles may be synthesizedfrom a variety of available polymers including but not limited topolylactide—polyglycolide copolymers, polyacrylates, polycaprolactones,albumin, gelatin, alginate, collagen, chitosan, polylactides, and poly(DL-lactide-co-glycolide) polymers. Nanoparticles may also includesilica nanoparticles, quantum dots, metal nanoparticles (e.g., gold, Cd,Se, ZnS, or iron oxide), or lanthanide nanoparticles.

In one embodiment of the present invention an embodiment of thecomposition or therapeutic agent (e.g., any of the agents or compoundsdisclosed herein) that up-regulates the sulfonating capacity of hairbearing skin is delivered as an implant that can be either passive oractive. In one embodiment, the passive drug delivery system can tunedrug release from the reservoir by controlling rates of diffusion,osmosis, or concentration gradients. In one embodiment, an active drugdelivery system (implant) can control drug release using a pump that canbe activated by a number of methods ranging from simple manual actuationfrom physical pressure to electrochemically driven mechanisms that canvary drug delivery rates.

In one embodiment of the present invention an embodiment of thecomposition or therapeutic agent (e.g., any of the agents or compoundsdisclosed herein) that up-regulates the sulfonating capacity of hairbearing skin is delivered as a slow releasing suspension, liposome, insitu gel-forming system, microsphere, non-aqueous solution/suspension,or implant.

In one embodiment of the present invention an embodiment of thecomposition or therapeutic agent (e.g., any of the agents or compoundsdisclosed herein) that up-regulates the sulfonating capacity of hairbearing skin is delivered as a slow releasing injectable or implantdaily, weekly, monthly, or quarterly.

In one embodiment of the present invention an embodiment of thecomposition or therapeutic agent (e.g., any of the agents or compoundsdisclosed herein) that up-regulates the sulfonating capacity of hairbearing skin is prepared as a slow releasing agent by acylation (albuminbinder), carbohydrate analogue attachment, poly amino acid fusion,PEGylation, Albumin or Fc Fusion (FcRn recycling).

In yet another embodiment of the present invention, minoxidil and anembodiment of the composition or therapeutic agent (e.g., any of theagents or compounds disclosed herein) that up-regulates the sulfonatingcapacity of hair bearing skin are delivered together as a slow releasinginjectable or implant.

In yet another embodiment minoxidil and an embodiment of the compositionor therapeutic agent (e.g., any of the agents or compounds disclosedherein) that up-regulates the sulfonating capacity of hair bearing skinare delivered together as a microparticle-based depot formulation,nanoparticle-based depot formulation, transdermal system, or implant.

Embodiments of the treatments disclosed herein can involveadministration of an embodiment of the compositions, activators,inducers, inhibitors, and/or agents disclosed herein for up-regulatingthe sulfonating capacity of hair bearing skin, hair follicles, and/orkeratinocyte cells, increasing minoxidil response, converting minoxidilnon-responders to responders, and/or accelerating minoxidil response.

One skilled in the art will appreciate with the benefit of the presentdisclosure that any one or combination of the treatment methodsdisclosed herein can be used in combination with any other treatmentmethod. In addition, treatment methods can be used in variouspermutations. For instance, a treatment method can involveadministration of a first composition selected to: up-regulate thesulfonating capacity of hair bearing skin, hair follicles, and/orkeratinocyte cells; increase minoxidil response; convert minoxidilnon-responders to responders; and/or accelerate minoxidil response by afirst technique (e.g., inhibiting Keap1) and then a subsequentadministration of a second composition selected to: up-regulate thesulfonating capacity of hair bearing skin, hair follicles, and/orkeratinocyte cells; increase minoxidil response; convert minoxidilnon-responders to responders; and/or accelerate minoxidil response by asecond technique (e.g., increasing the activity of p300/CBP). As anon-limiting example, a treatment method can involve administration of afirst composition including a Keap1 inhibitor agent for a first periodof time (e.g., 1 week, 1 month, etc.), followed by an application of asecond composition including an agent for increasing the activity ofp300/CBP for a second period of time (e.g., 1 week, 1 month, etc.). Oneskilled in the art can appreciate that other treatment methods, periodsof time, frequencies of administration, methods of administration, etc.can be used as variables in the combinations and permutations that makeup the overall treatment method.

This disclosure uses the terms optical density (OD) and absprbance units(AU) interchangeably.

The various methods and techniques described above provide a number ofways to carry out the invention. Of course, it is to be understood thatnot necessarily all objectives or advantages described can be achievedin accordance with any particular embodiment described herein. Thus, forexample, those skilled in the art will recognize that the methods can beperformed in a manner that achieves or optimizes one advantage or groupof advantages as taught herein without necessarily achieving otherobjectives or advantages as taught or suggested herein. A variety ofalternatives are mentioned herein. It is to be understood that someembodiments specifically include one, another, or several features,while others specifically exclude one, another, or several features,while still others mitigate a particular feature by inclusion of one,another, or several advantageous features.

Furthermore, the skilled artisan will recognize the applicability ofvarious features from different embodiments. Similarly, the variouselements, features and steps discussed above, as well as other knownequivalents for each such element, feature or step, can be employed invarious combinations by one of ordinary skill in this art to performmethods in accordance with the principles described herein. Among thevarious elements, features, and steps some will be specifically includedand others specifically excluded in diverse embodiments.

Although the application has been disclosed in the context of certainembodiments and examples, it will be understood by those skilled in theart that the embodiments of the application extend beyond thespecifically disclosed embodiments to other alternative embodimentsand/or uses and modifications and equivalents thereof.

The recitation of ranges of values herein is merely intended to serve asa shorthand method of referring individually to each separate valuefalling within the range. Unless otherwise indicated herein, eachindividual value is incorporated into the specification as if it wereindividually recited herein. All methods described herein can beperformed in any suitable order unless otherwise indicated herein orotherwise clearly contradicted by context. The use of any and allexamples, or exemplary language (for example, “such as”) provided withrespect to certain embodiments herein is intended merely to betterilluminate the application and does not pose a limitation on the scopeof the application otherwise claimed. No language in the specificationshould be construed as indicating any non-claimed element essential tothe practice of the application.

Certain embodiments of this application are described herein. Variationson those embodiments will become apparent to those of ordinary skill inthe art upon reading the foregoing description. It is contemplated thatskilled artisans can employ such variations as appropriate, and theapplication can be practiced otherwise than specifically describedherein. Accordingly, many embodiments of this application include allmodifications and equivalents of the subject matter recited in theclaims appended hereto as permitted by applicable law. Moreover, anycombination of the above-described elements in all possible variationsthereof is encompassed by the application unless otherwise indicatedherein or otherwise clearly contradicted by context.

All patents, patent applications, publications of patent applications,and other material, such as articles, books, specifications,publications, documents, things, and/or the like, referenced herein arehereby incorporated herein by this reference in their entirety for allpurposes, excepting any prosecution file history associated with same,any of same that is inconsistent with or in conflict with the presentdocument, or any of same that can have a limiting affect as to thebroadest scope of the claims now or later associated with the presentdocument. By way of example, should there be any inconsistency orconflict between the description, definition, and/or the use of a termassociated with any of the incorporated material and that associatedwith the present document, the description, definition, and/or the useof the term in the present document shall prevail.

EXAMPLES Example 1: In-Vitro Evaluation of SulfotransferaseUp-Regulation

The following experiment was conducted to assess the up-regulation ofsulfotransferase in hair follicles by candidate compounds know tointeract with human nuclear receptors (e.g., PXR and CAR).

Method: For each compound tested, 24 hairs were plucked from a humansubject. Hairs were visually inspected to confirm the presence of thehair bulb. 2 hairs were placed in to 12 clean sample tubes (i.e., twohairs per reaction) containing 100 uL of Williams' E media with 0.292g/L L-glutamine. Suitable candidate compounds were dissolved in DMSOsuch that when 1 uL of the compound solution was added to the samplemedia the final concentration of the compound was the concentrationbeing studied (listed in the Results section). 1 uL of DMSO with nocompound was added to control samples. Hairs were allowed to incubatewith the candidate compounds for 24 h at room temperature. All sampleswere run in triplicate, i.e., the data describe in the result section isan average of three samples tested.

After 24h of incubation hairs were removed from the sample media andwashed briefly with clean water. Hairs were trimmed to a length of ˜1 cmand immersed, bulb first, in 100 μL of an assay solution containing 50mM phosphate buffer (pH8), 5 mM potassium p-nitrophenyl sulfate, 20 μMadenosine 3′,5′-diphosphate, 100 μM minoxidil and 5 mM MgCl2. Hairs wereallowed to react with the solution for 24 hours at room temperature.After incubation, hairs were removed and the optical absorbance of thesolution at 405 nm was determined with a spectrophotometer using asingle scan and 1 cm path length.

Results:

TABLE 1 Up-Regulation of Sulfotransferase Via Interaction With HumanNuclear Receptors AB-001 Time (hrs): 24 Patient: Control ODs: OD 405 (1)OD 405 (2) OD 405 (3) Average STDEV 0.121 0.111 0.127 0.120 0.008 DrugsVitamin K2 OD 405 (1) OD 405 (2) OD 405 (3) Average STDEV  5 uM 0.1760.211 0.173 0.187 0.021 10 uM 0.339 0.138 0.180 0.219 0.106 20 uM 0.1450.208 0.144 0.166 0.037 AB-001 Time (hrs): 24 Patient: Control ODs: OD405 (1) OD 405 (2) OD 405 (3) Average STDEV 0.118 0.337 0.267 0.2410.112 Drugs Genistein OD 405 (1) OD 405 (2) OD 405 (3) Average STDEV 10uM 0.186 0.235 0.134 0.185 0.051 25 uM 0.179 0.144 0.085 0.136 0.048 50uM 0.122 0.080 0.190 0.131 0.056

TABLE 2 Up-Regulation of Sulfotransferase Via Interaction With HumanNuclear Receptors AB-001 Time (hrs): 24 Patient: Control ODs: OD 405 (1)OD 405 (2) OD 405 (3) Average STDEV 0.273 0.132 0.179 0.195 0.072 DrugsHyperforin OD 405 (1) OD 405 (2) OD 405 (3) Average STDEV 0.2 uM 0.4110.360 0.298 0.356 0.057   1 uM 0.475 0.402 0.369 0.415 0.054   5 uM0.523 0.506 0.489 0.506 0.017

Conclusions: Hyperforin, genistein, and vitamin K2 all inducedsulfotransferase activity in plucked hair follicles.

Example 2

A study was conducted on thirty human subjects (30 men) with lowsulfotransferase activity as determined by colorimetric enzymatic testdescribed by Goren et al. Subjects were recruited based on an enzymatictest result of OD<0.4 The subjects were randomized to an active groupand a placebo group in a 1:1 ratio. All subjects applied 5% topicalminoxidil once daily. The active group applied a shampoo containinghyperforin once a day prior to the use of minoxidil. The placebo groupapplied a vehicle shampoo. At the end of 1 week the averagesulfotransferase activity of all subjects increased almost 3 fold(287%). The average sulfotransferase activity remained high for afollow-up period of 6 months. At 4 months, the average increase in haircounts in the active group was 166% higher than the placebo. Inaddition, 60% of the subjects in the active group responded to minoxidilcompared to 0% of the placebo group. This demonstrates that embodimentsof the method and compositions disclosed herein can convert androgeneticalopecia pateints who are non-responders to minoxidil into responders.

Example 3

A study was conducted on thirty human subjects (30 women) with lowsulfotransferase activity as determined by colorometirc enzymatic testdescribed by Goren et al. Subjects were recruited based on an enzymatictest result of OD<0.4 The subjects were randomized to an active groupand a placebo group in a 1:1 ratio. All subjects applied 5% topicalminoxidil once daily. The active group applied a shampoo containinghyperforin once a day prior to the use of minoxidil. The placebo groupapplied a vehicle shampoo. At the end of 1 week the averagesulfotransferase activity of all subjects increased almost 3 fold(279%). The average sulfotransferase activity remained high for afollow-up period of 6 months. At 6 months, the average increase in haircounts in the active group was 151% higher than the placebo. Inaddition, 60% of the subjects in the active group responded to minoxidilcompared to 0% of the placebo group. This demonstrates that embodimentsof the method and compositions disclosed herein can convert androgeneticalopecia pateints who are non-responders to minoxidil into responders.

Example 4

A study was conducted on thirty human subjects (30 men) with lowsulfotransferase activity as determined by colorometirc enzymatic testdescribed by Goren et al. Subjects were recruited based on an enzymatictest result of OD<0.4 The subjects were randomized to an active groupand a placebo group in a 1:1 ratio. All subjects applied 5% topicalminoxidil once daily. The active group applied a shampoo containing St.John Wort once a day prior to the use of minoxidil. The placebo groupapplied a vehicle shampoo. At the end of 1 week the averagesulfotransferase activity of all subjects increased almost 3 fold(175%). The average sulfotransferase activity remained high for afollow-up period of 6 months. At 4 months, the average increase in haircounts in the active group was 123% higher than the placebo. Inaddition, 60% of the subjects in the active group responded to minoxidilcompared to 0% of the placebo group. This demonstrates that embodimentsof the method and compositions disclosed herein can convert androgeneticalopecia pateints who are non-responders to minoxidil into responders.

Example 5

A study was conducted on thirty human subjects (30 women) with lowsulfotransferase activity as determined by colorometirc enzymatic testdescribed by Goren et al. Subjects were recruited based on an enzymatictest result of OD<0.4 The subjects were randomized to an active groupand a placebo group in a 1:1 ratio. All subjects applied 5% topicalminoxidil once daily. The active group applied a shampoo containing St.John Wort once a day prior to the use of minoxidil. The placebo groupapplied a vehicle shampoo. At the end of 1 week the averagesulfotransferase activity of all subjects increased almost 3 fold(302%). The average sulfotransferase activity remained high for afollow-up period of 6 months. At 6 months, the average increase in haircounts in the active group was 142% higher than the placebo. Inaddition, 60% of the subjects in the active group responded to minoxidilcompared to 0% of the placebo group. This demonstrates that embodimentsof the method and compositions disclosed herein can convert androgeneticalopecia pateints who are non-responders to minoxidil into responders.

Example 6

A study was conducted to evaluate the efficacy of AB-103 (AB-103 formulais a combination of RXR+NR (nuclear receptors)) as an adjuvant therapyto 5% topical minoxidil solution in the treatment of Male Pattern HairLoss (MPHL).

Methodology: Double blinded, head-to-head prospective study.

Number of Patients: 48 male subjects

Diagnosis and Inclusion Criteria:

-   -   Subject diagnosed by a dermatologist with male pattern hair loss        (AGA)    -   Age: 18 and above    -   Subjects are able to give informed consent

Test product: AB-103 daily shampoo administered concomitantly withtopical 5% minoxidil solution, b.i.d.

Duration of treatment: 16 weeks

Reference therapy, dose and mode of administration: Vehicle shampooadministered concomitantly with topical 5% minoxidil solution, b.i.d.

Criteria for Evaluation:

Efficacy:

-   -   Each subject's global photographs before and after treatment        were assessed for hair growth by a blinded expert. The        assessment was made using a standardized 7 point rating scale        [baseline, week 8, week 16].    -   The activity of the sulfotransferase enzyme from each subject's        plucked hair was measured utilizing the Minoxidil Response Test        [baseline, week 1, week 16].

Safety:

-   -   Each subject's scalp was evaluated by the site investigator for        irritation, sensitization, erythema or any other abnormal        dermatological finding [baseline, week 1, week 8, week 16].

Statistical methods: The statistical method chosen to describe the datais the Mann-Whitney U test.

Summary—Conclusions:

Efficacy Results

The aim of the study was to evaluate the efficacy of AB-103 as anadjuvant therapy to 5% topical minoxidil solution in the treatment ofMPHL. A head-to-head prospective clinical study of 48 subjects treatedfor a minimum of 16 weeks was conducted. Subjects were randomized to twotreatment arms: AB-103 administered concomitantly with topical minoxidil(treatment arm) or a vehicle shampoo administered concomitantly withtopical minoxidil (comparator arm). Global photographers were evaluatedby a blinded expert. The treatment response was rated on a 7 point scaleas follows: −3 (significantly worse), −2 (moderately worse), −1(slightly worse), 0 (no change), +1 (slightly improved), +2 (moderatelyimproved) and +3 (significantly improved). The raw data from the CRFswas tabulated and graphed by an expert (refer to FIG. 1).

Eight subjects from the treatment arm and five subjects from thecomparator arm did not complete the study. No reason was given fordiscontinuation; however, at the one week post discontinuation follow-upcall none of these subjects reported adverse events. In total, 35subjects completed the study.

The Mann-Whitney U test (MedCalc v18.2.1) was used to determine theeffect of AB-103 as an adjuvant minoxidil therapy in the treatment armversus the vehicle shampoo in the comparator arm. The null and alternatehypothesis are given below:

-   -   H₀: The distributions of the subjects' treatment response in the        treatment arm and the comparator arm are equal    -   H_(A): The distributions of the subjects' treatment response in        the treatment arm and the comparator arm are not equal

The results indicate the following:

-   -   Mann-Whitney U test (two-sided)        -   Mann-Whitney U=93.5, p-value=0.0432; thus, the null            hypothesis is rejected.        -   Safety Results: No adverse events were reported in either            arm.

Conclusion:

The distributions of the subjects' treatment response in the AB-103+minoxidil arm and the vehicle shampoo+minoxidil arm were significantlydifferent (p-value=0.0432). In the AB-103+minoxidil arm 75% of subjectsexperienced hair growth versus 48% in the vehicle shampoo+minoxidil arm.

Note: In this sample population, the rate of response in the comparatorarm (48%) was larger than one would expect from prior clinical studies(39%). While it is possible that the vehicle improved some subjectsresponse, it is more likely that the higher response rate observed isdue to the study's small population size.

Example 7

A study was conducted to evaluate the efficacy of AB-103 inup-regulating the minoxidil sulfotransferase enzymes in human hairfollicles in-vivo.

Methodology: Double blinded, placebo controlled prospective study.

Number of Patients: 20 female subjects

Diagnosis and Inclusion Criteria:

-   -   Subject diagnosed by a dermatologist with female pattern hair        loss    -   Age: 18 and above    -   Subjects are able to give informed consent

Test product: AB-103 daily washout shampoo, q.d.

Duration of treatment: 7 days

Reference therapy, dose and mode of administration: Vehicle shampoo(Placebo), q.d.

Criteria for Evaluation:

Efficacy:

-   -   The activity of the sulfotransferase enzyme from each subject's        plucked hair was measured utilizing the Minoxidil Response Test        [baseline, day 7].

Safety:

-   -   Each subject's scalp was evaluated by the site investigator for        irritation, sensitization, erythema or any other abnormal        dermatological finding [baseline, day 7].

Statistical Methods:

The statistical method chosen to describe the data is the independentsamples t-test.

Summary—Conclusions:

Efficacy Results

The aim of the study was to evaluate the efficacy of AB-103 inup-regulating the minoxidil sulfotransferase enzymes in human hairfollicles in-vivo. A double blinded, placebo controlled prospectiveclinical study of 20 subjects treated for 7 days was conducted. Subjectswere randomized to two treatment groups: AB-103 shampoo or the shampoovehicle. Sulfotransferase activity levels were determined utilizing theMinoxidil Response Test. The analysis was conducted with aspectrophotometer (Shimadzu UV-1700, Kyoto, Japan). The raw data fromthe CRFs was tabulated and graphed by an expert (refer to FIG. 2).

The independent samples t-test (MedCalc v18.2.1) was used to determinethe effect of AB-103 compared to the vehicle shampoo on follicularsulfotrasnferase activity. The null and alternate hypothesis are givenbelow:

-   -   H₀: The mean change in sulfotrasnferase activity after 7 days of        treatment with AB-103 is equal to the mean sulfotrasnferase        activity after 7 days of treatment with the vehicle shampoo    -   H_(A): The mean change in sulfotrasnferase activity after 7 days        of treatment with AB-103 is greater then the mean        sulfotrasnferase activity after 7 days of treatment with the        vehicle shampoo    -   Mathematically written as:

H ₀:μ1=μ2

H _(A):μ1>μ2

The results indicate the following:

-   -   Independent samples t-test (two-sided)        -   The D'Agostino-Pearson test for normal distribution (MedCalc            v18.2.1) accepts normality of the differences with p=0.4278.        -   The analysis of the dataset yielded t(18)=−2.839, p=0.0109;            therefore, the results of t-test indicate that the null            hypothesis is rejected and we conclude that there was a            significant difference in the mean change in            sulfotransferase enzyme activity between the AB-103 group            (μ=0.2413, sd=−0.2562) [95% CI: 0.05807 to 0.4246] and the            placebo group (μ=−0.008420, sd=−0.1085) [95% CI: −0.08602 to            0.06918].        -   The 95% CI for the difference in means is            0.06492≤μ_(b)−μ_(a)≤0.4346 with the best estimate being the            mean difference=0.2497.    -   Safety Results: No adverse events were reported in either group.

Conclusion:

The mean change in sulfotrasnferase activity after 7 days of treatmentwith AB-103 was significantly greater than the mean change followingplacebo treatment (p-value<0.0109). In addition, utilizing the data fromreport (FI-IVD-001) regulatory submission, we conclude that the increasein sulfotrasnferase activity was significantly larger then thewithin-subject variability of the MRT assay.

Example 8

A study was conducted to evaluate the efficacy of AB-103 as an adjuvanttherapy to 5% topical minoxidil foam in the treatment of Female PatternHair Loss (FPHL).

Methodology: Double blinded, head-to-head prospective study.

Number of Patients: 30 female subjects

Diagnosis and Inclusion Criteria:

-   -   Subject diagnosed by a dermatologist with female pattern hair        loss    -   Age: 18 and above    -   Subjects are able to give informed consent

Test product: AB-103 daily washout shampoo administered concomitantlywith topical 5% minoxidil foam, q.d.

Duration of treatment: 24 weeks

Reference therapy, dose and mode of administration: Topical 5% minoxidilfoam, q.d.

Criteria for Evaluation:

Efficacy:

-   -   Each subject's global photographs before and after treatment        were assessed for hair growth by a blinded expert. The        assessment was made using a standardized 7 point rating scale        [baseline, week 12, week 24].    -   The activity of the sulfotransferase enzyme from each subject's        plucked hair was measured utilizing the Minoxidil Response Test        [baseline, week 1, week 24].

Safety:

-   -   Each subject's scalp was evaluated by the site investigator for        irritation, sensitization, erythema or any other abnormal        dermatological finding [baseline, week 4, week 8, week 12, week        24].

Statistical Methods:

The statistical method chosen to describe the data is the Mann-Whitney Utest.

Summary—Conclusions:

Efficacy Results

The aim of the study was to evaluate the efficacy of AB-103 as anadjuvant therapy to 5% topical minoxidil foam in the treatment of FPHL.A head-to-head prospective clinical study of 30 subjects treated for aminimum of 24 weeks was conducted. Subjects were randomized to twotreatment groups: AB-103 administered concomitantly with topicalminoxidil or topical minoxidil mono-therapy. Global photographers wereevaluated by a blinded expert. The treatment response was rated on a 7point scale as follows: −3 (significantly worse), −2 (moderately worse),−1 (slightly worse), 0 (no change), +1 (slightly improved), +2(moderately improved) and +3 (significantly improved). The raw data fromthe CRFs was tabulated and graphed by an expert (refer to FIG. 3).

The Mann-Whitney U test (MedCalc v18.2.1) was used to determine theeffect of AB-103+minoxidil treatment versus minoxidil mono-therapy. Thenull and alternate hypothesis are given below:

-   -   H₀: The distributions of the AB-103+minoxidil group and the        minoxidil mono-therapy group are equal    -   H_(A): The distributions of the AB-103+minoxidil group and the        minoxidil mono-therapy group are not equal

The results indicate the following:

-   -   Mann-Whitney U test (two-sided)        -   Combined Dataset (all sites): Mann-Whitney U test            p-value<0.0357; thus, the null hypothesis is rejected.        -   Safety Results: No adverse events were reported in either            group.

Conclusion:

The treatment difference between AB-103+minoxidil and minoxidilmono-therapy was statistically significant (p-value<0.0357). In theAB-103+minoxidil group 66% of subjects experienced hair growth versus33% in the minoxidil mono-therapy group.

Albeit the small study population, this study provide initial supportfor the use of AB-103 as an adjuvant therapy to 5% topical minoxidilfoam in the treatment of FPHL.

Example 9

A study was to evaluate the efficacy of AB-103 as an adjuvant therapy to5% topical minoxidil foam in the treatment of FPHL patientnon-responders to 5% topical minoxidil.

Methodology: Double blinded, placebo controlled prospective study.

Number of Patients: 30 female subjects

Diagnosis and Inclusion Criteria:

-   -   Subject diagnosed by a dermatologist with female pattern hair        loss    -   Age: 18 and above    -   Subjects had a negative Minoxidil Response Test result (OD<0.4)    -   Subjects are able to give informed consent

Test product: AB-103 daily washout shampoo administered concomitantlywith topical 5% minoxidil foam, q.d.

Duration of treatment: 24 weeks

Reference therapy, dose and mode of administration: Vehicle shampooadministered concomitantly with topical 5% minoxidil foam, q.d.

Criteria for Evaluation:

Efficacy:

-   -   Mean change in target area hair counts (TAHC) [baseline, week        24].    -   The activity of the sulfotransferase enzyme from each subject's        plucked hair was measured utilizing the Minoxidil Response Test        [baseline, week 1, week 24].

Safety:

-   -   Each subject's scalp was evaluated by the site investigator for        irritation, sensitization, erythema or any other abnormal        dermatological finding [baseline, week 24].

Statistical Methods:

The statistical method chosen to describe the data is the independentsamples t-test.

Summary—Conclusions:

Efficacy Results

The aim of the study was to evaluate the efficacy of AB-103 as anadjuvant therapy to 5% topical minoxidil in the treatment of FPHLpatients non-responders to 5% topical minoxidil. A double blindedplacebo controlled prospective clinical study of 30 subjects treated fora minimum of 24 weeks was conducted. Subjects were randomized to twotreatment groups: AB-103 administered concomitantly with topicalminoxidil or a vehicle shampoo administered concomitantly with topicalminoxidil (placebo group). TAHC was evaluated in a shaved and tattooed 1cm² area of the scalp. The data from the CRFs was tabulated and graphedby an expert (refer to FIG. 4). Three subjects from the placebo groupdid not complete the study and one subject from the treatment group didnot complete the study. No adverse events were reported in thesesubjects. Two of the subjects that discounted the study cited lack ofvisible results as a reason for discontinuation. In total, 26 subjectscompleted the study.

The independent samples t-test (MedCalc v18.2.1) was used to determinethe mean change in TAHC in the AB-103+minoxidil group compared to themean change in TAHC in the vehicle shampoo+minoxidil group. The null andalternate hypothesis are given below:

-   -   H₀: The mean change in TAHC after 24 weeks of treatment with        AB-103+minoxidil is equal to the mean change in TAHC after 24        weeks of treatment with the vehicle shampoo+minoxidil    -   H_(A): The mean change in TAHC after 24 weeks of treatment with        AB-103+minoxidil is greater than the mean change in TAHC after        24 weeks of treatment with the vehicle shampoo+minoxidil    -   Mathematically written as:

H ₀:μ1=μ2

H _(A):μ1>μ2

The results indicate the following:

Independent Samples t-Test (Two-Sided)

-   -   The D'Agostino-Pearson test for normal distribution (MedCalc        v18.2.1) accepts normality of the differences with p=0.4063.    -   The analysis of the dataset yielded t(24)=−2.862, p=0.0086;        therefore, the results of t-test indicate that the null        hypothesis is rejected and we conclude that there was a        significant difference in the mean change in TAHC between the        AB-103 group (μ=12.9571, sd=9.4802) [95% CI: 7.4835 to 18.4308]        and the placebo group (μ=3.0083, sd=8.0062) [95% CI: −2.0786 to        8.0952].    -   The 95% CI for the difference in means is        −17.1224≤μ_(b)−μ_(a)≤−2.7752 with the best estimate being the        mean difference=−9.9488.

Safety Results: No adverse events were reported in either group.

Conclusion:

The mean change in TAHC following 24 weeks of treatment withAB-103+minoxidil was greater than the mean change in TAHC in the placebogroup (p=0.0086). Further, in the AB-103+minoxidil group 57% of subjectsexperienced hair growth versus 0% in the placebo group.

This study provides support for the use of AB-103 as an adjuvant therapyto 5% topical minoxidil in the treatment of FPHL patient non-respondersto 5% topical minoxidil.

Example 10

Assessing the Efficacy of Alkaline pH on Increasing CollicularSulfotransferase.

Study 001

An in-vitro study was conducted to assess the efficacy of an alkaline pHsolution on increasing follicular sulfotransferase. Twenty hairs wereplucked from each subject. The baseline sulfotransferase activity ofeach subject was measured by analyzing 10 plucked hairs with anMinoxidil Response Test devised by Goren et al. The remaining 10 hairswere incubated with an alkaline solution (pH of 8.5) for a period of 24hour at a temperature of 37° C. Thereafter, the remaining 10 pluckedhairs were analyzed with the Minoxidil Response Test devised by Goren etal. The data is summarized in Table 3 below. The average increase wasapproximately 100%.

TABLE 3 Efficacy of Alkaline pH on Increasing CollicularSulfotransferase Baseline Post Treatment Subject Activity (OD) Activity(OD) 1 0.19 0.374 2 0.87 1.803 3 0.32 0.521

Study 002

A study was conducted on thirty human subjects (30 men) with lowsulfotransferase activity as determined by colorimetric enzymatic testdescribed by Goren et al. Subjects were recruited based on an enzymatictest result of OD<0.4. The subjects were randomized to an active groupand a placebo group in a 1:1 ratio. All subjects applied 5% topicalminoxidil once daily. The active group applied an alkaline solution (pHof 8.5) once a day prior to the use of minoxidil. The placebo groupapplied a vehicle solution (pH 7.0). At the end of 1 week, the averagesulfotransferase activity of all subjects increased almost 2 fold (95%).The average sulfotransferase activity remained high for a follow-upperiod of 6 months. At 4 months, the average increase in hair counts inthe active group was 192% higher than the placebo. In addition,approximately 50% of the subjects in the active group responded tominoxidil compared to 0% of the placebo group.

Study 003

A study was conducted on thirty human subjects (30 women) with lowsulfotransferase activity as determined by colorometirc enzymatic testdescribed by Goren et al. Subjects were recruited based on an enzymatictest result of OD<0.4. The subjects were randomized to an active groupand a placebo group in a 1:1 ratio. All subjects applied 5% topicalminoxidil once daily. The active group applied an alkaline solution (pHof 8.5) once a day prior to the use of minoxidil. The placebo groupapplied a vehicle solution (pH 7.0). At the end of 1 week, the averagesulfotransferase activity of all subjects increased almost 2 fold(115%). The average sulfotransferase activity remained high for afollow-up period of 6 months. At 6 months, the average increase in haircounts in the active group was 173% higher than the placebo. Inaddition, approximately 50% of the subjects in the active groupresponded to minoxidil compared to 0% of the placebo group.

Example 11

pHi Assay

Bromothymol Blue, 0.04% Aqueous solution was used as a pH indicator tovisualize intracellular pH (pHi) of cells located in the Hair FollicleStem Cell (HFSC) niche. In a neutral solution of pH 6.0-7.6 BromothylmolBlue appears green, below pH 6.0 it appears yellow, and above pH 7.6 itappears blue. Hair follicles were submerged in assay solution forapproximately 5 minutes, until HFSCs were stained. Cells were visualizedusing a 20× magnification stereo microscope equipped with a 5MP ColorDigital Eyepiece Microscope Camera.

TABLE 4 Composition/Information on Ingredients Chemical Name CAS # %EINICS Water 7732-18-5 99.96% 231-791-2 Bromothymol blue, 34722-90-2 0.04% 252-269-7 sodium salt

pHi of HFSC

The following experiment was conducted to assess the intracellular pH ofthe HFSC niche in hair.

Method:

1 hair was plucked from each human subject. Hairs were visuallyinspected to confirm the presence of the hair bulb and hairs were rinsedbriefly with clean water. Hairs were then immersed, bulb first, in 100μL of assay solution. Hairs were allowed to react with the solution for5 minutes at room temperature. After incubation, hairs were removed andmounted on a clean glass slide and visualized using a 20× magnificationstereo microscope equipped with a 5MP Color Digital Eyepiece MicroscopeCamera.

Results: See Table 5 of FIG. 5.

Conclusion: the intracellular pH of cells contained in the HFSC nichewas below pH 6.0

In Vitro Upregulation of SULT1A1 by Increasing pH (24h)

The following experiment was conducted to assess the up-regulation ofsulfotransferase in hair follicles by an increase in pHi.

Method:

For each pH tested, 36 hairs were plucked from a human subject. Hairswere visually inspected to confirm the presence of the hair bulb. 2hairs were placed in to 9 wells (i.e., two hairs per reaction) of asterile flat bottom tissue culture plate containing 250 uL of Williams'Emedia with 0.292 g/L L-glutamine at pH 5.9, 7.4, and 8.1 (i.e. 3 wellsper pH). Hairs were incubated for 6 hours and 24 hours at 5% CO₂, 100%relative humidity. All samples were run in triplicate, i.e. the datadescribed in the results section is an average of three samples testedfor each pH.

After 24 hours of incubation, hairs were removed from the sample mediaand washed briefly with clean water. Hairs were trimmed to a length of˜1 cm and immersed, bulb first, in 100 μL of an assay solutioncontaining 50 mM phosphate buffer (pH8), 5 mM potassium p-nitrophenylsulfate, 20 μM adenosine 3′,5′-diphosphate, 100 μM minoxidil and 5 mMMgCl2. Hairs were allowed to react with the solution for 24 hours atroom temperature. After incubation, hairs were removed and the opticalabsorbance of the solution at 405 nm was determined with aspectrophotometer using a single scan and 1 cm path length.

Results: See Tables 6-7 below.

TABLE 6 Incubation in media for 24 hour. OD 405 nm OD 405 nm OD 405 nmSubject pH 5.9 pH 7.4 pH 8.1 AB-011 0.113 0.118 0.115 AB-012 0.088 0.0740.080 AB-013 0.398 0.406 0.393 AB-014 0.444 0.423 0.446 AB-015 0.6120.622 0.626

TABLE 7 Incubation in media for 6 hours. OD 405 nm OD 405 nm OD 405 nmSubject pH 5.9 pH 7.4 pH 8.1 AB-011 0.102 0.233 0.473 AB-012 0.054 0.2060.314 AB-013 0.403 0.492 0.827 AB-014 0.421 0.933 1.108 AB-015 0.6211.344 1.665

Conclusion: Incubating hairs in growth medium above pH 7.0 was able tosignificantly upregulate sulfotransferase response in 24 hours, but notin 6 hours.

In Vivo Change of HFSC Niche pHi

The following experiment was conducted to assess the ability to changeHFSC pHi by applying a topical solution to the scalp.

Method:

1 mL of 10% solution of sodium bicarbonate in Dimethyl sulfoxide (DMSO)was applied to the scalp of each subject in a small quarter-sized area.1 hair from within the area was plucked from each subject at time 0(zero), 15, and 30 min. Hairs were visually inspected to confirm thepresence of the hair bulb and were rinsed briefly with clean water.Hairs were then immersed, bulb first, in 100 μL of assay solution. Hairswere allowed to react with the solution for 5 minutes at roomtemperature. After incubation, hairs were removed and mounted on a cleanglass slide and visualized using a 20× magnification stereo microscopeequipped with a 5MP Color Digital Eyepiece Microscope Camera.

Results: See Tables 8-10 of FIGS. 6-8.

Conclusion: Topical application of 10% sodium bicarbonate in DMSO wasable to change the pHi of cells located in the HFSC niche within 30minutes of application.

Example 12

Sulfotransferase Up-Regulation with Alkalizing Agents in Human HairFollicle

Twenty-four (24) hairs were plucked from each human subject. Hairs werevisually inspected to confirm the presence of the hair bulb. 2 hairswere placed in to an open 1.5 mL tube (i.e., two hairs per reaction)containing 400 μL of Dulbecco's Modified Eagle's Medium/Nutrient MixtureF-12 Ham media with 0.365 g/L L-glutamine, 1.2 g/L NaHCO₃, 5.18 g/LCHES, pH 9.5. Hairs were incubated for 0 (baseline), 4, 8, 16, 20, and24 hours at 37° C., 100% relative humidity. All samples were run inquadruplicate and the experiment was repeated four times, i.e. the datadescribed in the results section is an average of four experiments withfour samples tested for each time point.

After each time point of incubation, hairs were removed from the samplemedia and washed briefly with clean water. Hairs were trimmed to alength of ˜1 cm and immersed, bulb first, in 100 μL of an assay solutioncontaining 50 mM phosphate buffer (pH8), 5 mM potassium p-nitrophenylsulfate, 20 μM adenosine 3′,5′-diphosphate, 100 μM minoxidil and 5 mMMgCl2. Hairs were allowed to react with the solution for 24 hours atroom temperature. Referring to FIG. 9, after incubation, hairs wereremoved and the optical absorbance of the solution at 405 nm wasdetermined with a spectrophotometer using a single scan and 1 cm pathlength.

TABLE 8 Results of sulfotransferase up-regulation with alkalizing agentsstudy Time exposed to AVG OD % increase from pH 9.5 media (hrs) (405 nm)baseline (0 hr) Subject 1 0 0.287 0.0 4 0.297 3.4 8 0.393 37.1 16 0.52382.2 20 0.564 96.5 24 0.649 126.3 Subject 2 0 0.984 0.0 4 1.022 3.8 81.145 16.4 16 1.430 45.3 20 1.621 64.6 24 1.630 65.6

Example 13

Alkalizing Topical Formulas with and without Minoxidil

Table 9 shows various topical formulations containing a salt ofminoxidil (e.g., alkalinizing salts of minoxidil) with and withoutMinoxidil that may be used with embodiments of the present invention.

TABLE 9 Topical formulations containing a salt of minoxidil IngredientF1 F2 F3 F4 F5 Minoxidil 0.00 5.00 5.00 5.00 0.00 Glycerol 18.00 18.0010.80 10.80 18.00 BHT 0.05 0.05 0.05 0.05 0.05 Ethanol 20.70 45.40 44.9047.40 19.70 Water 50.00 23.00 30.00 30.00 50.00 Labrasol 5.00 5.00 5.003.00 5.00 Lecithin 1.25 1.25 1.25 0.75 1.25 NaHCO₃ 5.00 2.30 3.00 3.005.00 Span 20 0.00 0.00 0.00 0.00 1.00 Total 100.00 100.00 100.00 100.00100.00

Example 14

Human Hair Follicle pH Changes Versus Time with Topical Application ofAlkalizing Topical Formulas

Various topical formulations were applied to the scalp of subjects.Hairs from the application site were plucked hourly over an 24-hourperiod. Hairs after each pluck were imaged to detect the pH changecaused by each formulation over the duration of the experiment. The pHiassay described in Example 11 was utilized to detect elevated pH. Hairswere plucked once every hour for 8 hours and then again the next day (24h).

TABLE 10 Image results detecting pH change for plucked hairs Formula F1Formula F2 Formula F3 Formula F4 Time Subject Subject Subject SubjectSubject Subject Subject Subject (h) 1 2 1 2 1 2 1 2 1 Blue Blue YellowYellow Blue Blue Blue Blue 2 Blue Blue Yellow Yellow Yellow Yellow BlueBlue 3 Blue Blue NA NA Yellow Yellow Blue Blue 4 Blue Blue NA NA NA NABlue Blue 5 Blue Blue NA NA NA NA Blue Yellow 6 Blue Yellow NA NA NA NABlue Yellow 7 Blue Yellow NA NA NA NA Yellow Yellow 8 Yellow Yellow NANA NA NA Yellow Yellow 24 Yellow Yellow NA NA NA NA Yellow Yellow

Example 15

Accelerated Stability of Liposomal Containing Solutions

Various topical liposomal formulation were prepared and monitored foroxidative and hydrolysis degradation. Two (2) verification batches(lots) of a liposomal containing formulation were created and stored atelevated temperature. During the course of the accelerated stabilitystudy, samples of each lot will be removed from the elevated temperaturevisually inspected for yellow color.

TABLE 11 Formula G1 (pH 9.5-10.5) NO. Ingredient INCI Designation %Weight 1 WATER WATER (AQUA) 85.44 2 TETRASODIUM TETRASODIUM 0.10 EDTAEDTA 3 SODIUM SODIUM 8.40 BICARBOATE BICARBONATE 4 Accanon-CC6 PEG-6CAPRYLIC/ 1.00 CAPRIC GLYCERIDE 5 Glycerol Glycerin 1.75 6 Phospholipon90G Phosphatidylcholine 2.00 7 Fragrance FRAGRANCE 0.06 8 Polysorbate 80Polysorbate 80 0.25 9 EUXYL PE 9010 Phenoxyethanol, 1.00Ethylhexylglycerin

TABLE 12 Formula G2 (pH 7.5-9.5) NO. Ingredient INCI Designation %Weight 1 WATER WATER (AQUA) 90.84 2 TETRASODIUM TETRASODIUM 0.10 EDTAEDTA 3 TRIS TRIS 3.00 PEG-6 CAPRYLIC/CAPRIC 4 Accanon-CC6 GLYCERIDE 1.005 Glycerol Glycerin 1.75 6 Phospholipon 90G Phosphatidylcholine 2.00 7Fragrance FRAGRANCE 0.06 8 Polysorbate 80 Polysorbate 80 0.25 9 EUXYL PE9010 Phenoxyethanol, 1.00 Ethylhexylglycerin

Subjecting test devices to extreme temperature conditions is intended tosimulate product degradation over time, but at an accelerated rate.Using the Arrhenius equation, shelf-life data can be extrapolated as apredictive measurement for product performance. The Arrhenius equationrelates chemical reaction rate (k) to absolute temperature (T):

d(Ink)/dT=ΔE _(a) /RT ²

Where E_(a) is the activation energy and R is the universal gasconstant.

TABLE 13 Arrhenius Model for Liposomal Containing Solution StabilityDays Required Days Required Days Required Treatment for 1 year for 1.5year for 2 year Temperature stability @ stability @ stability @ (° C.)23° C. 23° C. 23° C. 50 56.2 days 84.3 days 112.3 days 40 112.3 days168.6 days 224.6 days

Using the Arrhenius model at 20 kcal, the test formula would havesatisfactory performance theoretically for 1 years if specificationswere met after 56.2 days at 50° C., or 112.3 days at 40° C.

The purpose of stability testing at 50° C. and 40° C. is to generatedata to determine the shelf life of the product. The data is to be usedas a predictor of stability for the final product form.

Test Kit Storage Conditions

Test formulas will be stored at 50° C. and 40° C. (±2° C. for alltemperatures).

Interval Between Analyses

TABLE 14 Liposomal Containing Solution Accelerated Stability StudyStorage Temp Duration 50° C. Day 6, 14, 28, 42, 56, 70, 84, 98 40° C.Day 6, 14, 28, 70, 98, 126, 154, 182

Stability Criteria

The accelerated stability studies must initially pass the acceptancetargets detailed in Table 15.

Specifications

TABLE 15 Liposomal Containing Solution Specificity Release Testing andStability Result Specification Result Description (visual) DeterminationVisual Inspection White Pass Visual Inspection Slight Yellow/Off- PassWhite Visual Inspection Yellow Fail

Testing Schedule

0 6 14 28 42 56 70 84 98 126 154 182 50 ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ 40 ✓ ✓ ✓ ✓ ✓ ✓ ✓✓

Results

TABLE 16 Accelerated Stability Report for Formula G1 (pH 9.5-1.5)Formula G1 Days 0 6 14 28 42 56 70 84 98 126 154 182 50 C Pass Pass PassPass Fail Fail NA NA NA NA NA NA 40 C Pass Pass Pass Pass Pass Pass PassFail Fail NA NA NA

TABLE 17 Accelerated Stability Report for Formula G2 (pH 7.5-9 5)Formula G2 (pH 7.5-9.5) Days 0 6 14 28 42 56 70 84 98 126 154 182 50 CPass Pass Pass Pass Pass Pass Pass Pass Pass Pass Pass Pass 40 C PassPass Pass Pass Pass Pass Pass Pass Pass Pass Pass Pass

Example 16

In Vivo Evaluation of SULT1A1 Expression after Application of Formula G1and G2 (Liposomal Containing Solutions)

An in-vivo study was conducted to assess the efficacy of a liposomecontaining solution on increasing follicular sulfotransferase. 24 femalesubjects were recruited to the study. 10 hairs were plucked from eachsubject at baseline (day 0). The baseline sulfotransferase activity ofeach subject was measured by analyzing 10 plucked hairs with anMinoxidil Response Test devised by Goren et al. Subsequent tosulfotransferase baseline assessment each subject was instructed toapply a liposomal containing solution to their scalp once daily forseven days. 12 subjects applied Formula G1 (Table 11) and 12 subjectsapplied Formula G2 (Table 12)

After seven days of treatment, 10 plucked hairs from each subject wereanalyzed with the Minoxidil Response Test devised by Goren et al. Thedata is summarized in Table 18 below. The average increase wasapproximately 145% for Formula G1 and 135% for formula G2.

TABLE 18 Efficacy of Formula G1 and G2 on Increasing FollicularSulfotransferase Subject Formula Baseline Activity (OD) Post TreatmentActivity (OD) 1 G1 0.13 1.58 2 G1 0.25 0.71 3 G1 0.42 0.62 4 G1 0.490.92 5 G1 0.01 0.45 6 G1 0.09 0.36 7 G1 0.08 0.25 8 G1 0.43 0.41 9 G10.31 0.36 10 G1 0.36 0.89 11 G1 0.51 0.74 12 G1 0.01 0.29 13 G2 0.030.53 14 G2 0.24 1.23 15 G2 0.26 0.27 16 G2 0.39 0.58 17 G2 0.31 0.69 18G2 0.27 0.35 19 G2 0.26 0.82 20 G2 0.14 0.39 21 G2 0.19 0.46 22 G2 0.320.67 23 G2 0.59 0.86 24 G2 0.04 0.29

1. A composition for increasing the sulfonating capacity of a hairfollicle cell and/or keratinocyte cell, inducing sulfotransferase in ahair follicle, inducing expression of sulfotransferase, increasingsulfotransferase activity, upregulating sulfotransferase, convertingnon-responders to minoxidil responders, and/or improving minoxidilresponse, comprising: an agent configured to induce expression ofsulfotransferase in skin of a person and an alkalinizing agent, whereinthe alkalinizing agent is placed in a liposome solution containinglecithin or phosphatidylcholine.
 2. The composition of claim 1, furthercomprising minoxidil and/or a penetration enhancer.
 3. The compositionof claim 1, wherein the composition is configured to be administered byapplying the composition at a predetermined frequency and/oradministering the composition prior to an application of topicalminoxidil.
 4. The composition of claim 1, wherein the composition isformulated as a cosmetic product.
 5. The composition of claim 1, whereinthe composition is formulated as any one or combination of a timerelease vehicle, a cream, a solution, a lotion, a serum, an ointment, aspray, an aerosol medium, a capsule, a shampoo, a gel, a foam, acosmetic, a hair conditioner, a hair care product, a hair mask, adeodorant, an antiperspirant, a moisturizer, or a shaving cream or gel.6. The composition of claim 1, further comprising an inorganic sulfursource, wherein the inorganic sulfur source is chosen from the groupconsisting of cysteine, L-cysteine, hydrogen sulfide, elemental sulfur,sulfite, thiosulfate, polythionates, magnesium sulfate, sodium sulfate,and sodium metabisulfite.
 7. The composition of claim 1, wherein theliposome solution includes a buffering solution to stabilize the pH ofthe liposome solution.
 8. The composition of claim 7, wherein thebuffering solution includes sodium bicarbnate, acetate, citrate, Tris,or HEPES.
 9. The composition of claim 1, wherein the liposome solutionincludes an antioxidant to eliminate or slow oxidative degradation ofthe liposome solution.
 10. The composition of claim 9, wherein theantioxidant includes ascorbic acid, monothioglycerol, potassiummetabisulfite, sodium bisulfite, sodium formaldehyde sulfoxylate, sodiumsulfite, sodium thiosulfate, tocopherol, sodium metabisulfite, butylatedhydroxytoluene (BHT), butylated hydroxyanisole (BHA), ascorbylpalmitate, propyl gallate, acai oil, alpha lipoic acid, green teaextract, retinol, vitamin C, coenzyme Q10 (Co Q-10), isoflavones,polyphenols, curcumin, turmeric, pomegranate, rosemary extract,glutathione, selenium, zinc, a chelating molecule,ethylenediaminetetraacetic acid (EDTA), disodium EDTA, tetrasodium EDTA,pentasodium penetate, sodium metasilicate and phosphate derivatives,etidronic acid and/or its derivatives, and/or galactaric acid.
 11. A kitfor increasing the sulfonating capacity of a hair follicle cell and/orkeratinocyte cell, inducing sulfotransferase in a hair follicle,inducing expression of sulfotransferase, increasing sulfotransferaseactivity, upregulating sulfotransferase, converting non-responders tominoxidil responders, and/or improving minoxidil response, the kitcomprising a composition and a dispenser, implant, or pill; wherein thecomposition comprises: an agent configured to induce expression ofsulfotransferase in skin of a person and an alkalinizing agent, whereinthe alkalinizing agent is placed in a liposome solution containinglecithin or phosphatidylcholine.
 12. The kit of claim 11, wherein thecomposition is formulated as any one or combination of a time releasevehicle, a cream, a solution, a lotion, a serum, an ointment, a spray,an aerosol medium, a capsule, a shampoo, a gel, a foam, a cosmetic, ahair conditioner, a hair care product, a hair mask, a deodorant, anantiperspirant, a moisturizer, or a shaving cream or gel.
 13. The kit ofclaim 11, wherein the composition further includes minoxidil.
 14. Thekit of claim 11, wherein: the liposome solution includes a bufferingsolution to stabilize the pH of the liposome solution; or the liposomesolution includes an antioxidant to eliminate or slow oxidativedegradation of the liposome solution.
 15. A method of stabilizing acomposition, the method comprising: encapsulating a composition in asolution comprising liposome, non-ionic liposome, niosome, novasome I,erythromycin-Zn complex, microsphere, nanoparticle, solid lipidnanoparticle, nanoemulsion, micelle, nanogel, selenium nanoparticle,bilosome, dendrimer, or carbon nanotube; wherein the encapsulationincreases water solubility of the composition, decreases watersolubility of the composition, reduces loss through degradation, orpromotes penetration through the stratum corneum.
 16. The method ofclaim 15, wherein: the liposomal solution is stabilized via maintaininga pH level of the liposomal solution between pH of 7-11 by adding abuffering solution to the liposomal solution and/or eliminating orslowing oxidative degradation of the liposome solution by adding anantioxidant to the liposomal solution.
 17. The method of claim 16,wherein: the buffering solution includes sodum bicarbonate, acetate,citrate, Tris, or HEPES; the antioxidant includes ascorbic acid,monothioglycerol, potassium metabisulfite, sodium bisulfate, sodiumformaldehyde sulfoxylate, sodium sulfite, sodium thiosulfate,tocopherol, sodium metabisulfite, butylated hydroxytoluene (BHT),butylated hydroxyanisole (BHA), ascorbyl palmitate, propyl gallate, acaioil, alpha lipoic acid, green tea extract, retinol, vitamin C, coenzymeQ10 (Co Q-10), isoflavones, polyphenols, curcumin, turmeric,pomegranate, rosemary extract, glutathione, selenium, zinc, a chelatingmolecule, ethylenediaminetetraacetic acid (EDTA), disodium EDTA,tetrasodium EDTA, pentasodium penetate, sodium metasilicate andphosphate derivatives, etidronic acid and/or its derivatives, and/orgalactaric acid.
 18. The method of claim 17, further comprising:encapsulating sodium bicarbonate buffer or Tris buffer inphosphatidylcholine.
 19. The method of claim 15, wherein: the compoitionincludes an agent configured to induce expression of sulfotransferaseand an alkalinizing agent, wherein encapsulating the compositioninvolves placing the alkalinizing agent in the liposomal solution. 20.The method of claim 19, wherein: the liposomal solution containslecithin or phosphatidylcholine.